CA2671299A1 - Functions and targets of let-7 micro rnas - Google Patents

Abstract

The present invention concerns methods and compositions for treating or assessing treatment of diseases related to mis-expression of genes or genetic pathways that can be modulated by let-7. Methods may include evaluating patients for genes or genetic pathways modulated by let-7, and/or using an expression profile to assess the condition of a patient or treating the patient with an appropriate miRNA.

Description

DESCRIPTION

[0001] This application is related to U.S. Patent Applications serial number 11/141,707 filed May 31, 2005 and serial number 11/273,640 filed November 14, 2005, each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION
1. FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of molecular biology.
More particularly, it concerns methods and compositions involving diagnosis and treatment of disorders related to biologic pathways that are directly or indirectly modulated by the let-7 microRNA (miRNAs) family.

II. BACKGROUND

[0003] In 2001, several groups used a cloning method to isolate and identify a large group of "microRNAs" (miRNAs) from C. elegans, Drosophila, and humans (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Several hundreds of miRNAs have been identified in plants and animals - including humans -which do not appear to have endogenous siRNAs. Thus, while similar to siRNAs, miRNAs are distinct.

[0004] miRNAs thus far observed have been approximately 21-22 nucleotides in length and they arise from longer precursors, which are transcribed from non-protein-encoding genes. See review of Carrington et al. (2003). The precursors form structures that fold back on themselves in self-complementary regions; they are then processed by the nuclease Dicer in animals or DCLl in plants. miRNA molecules interrupt translation through precise or imprecise base-pairing with their targets.

[0005] Many miRNAs are conserved among diverse organisms, and this has led to the suggestion that miRNAs are involved in essential biological processes throughout the life span of an organism (Esquela-Kerscher and Slack, 2006). In particular, miRNAs have been implicated in regulating cell growth, and cell and tissue differentiation; cellular processes that are associated with the development of cancer.
For instance, lin-4 and let-7 both regulate passage from one larval state to another during C. elegans development (Ambros, 2001). mir-14 and bantam are Drosophila miRNAs that regulate cell death, apparently by regulating the expression of genes involved in apoptosis (Brennecke et al., 2003, Xu et al., 2003).

[0006] Research on miRNAs is increasing as scientists are beginning to appreciate the broad role that these molecules play in the regulation of eukaryotic gene expression. In particular, several recent studies have shown that expression levels of numerous miRNAs are associated with various cancers (reviewed in Esquela-Kerscher and Slack, 2006). Reduced expression of two miRNAs correlates strongly with chronic lymphocytic leukemia in humans, providing a possible link between miRNAs and cancer (Calin et al., 2002). Others have evaluated the expression patterns of large numbers of miRNAs in multiple human cancers and observed differential expression of almost all miRNAs across numerous cancer types (Lu et al., 2005). Most studies link miRNAs to cancer only by indirect evidence. However, He et al. (2005) has provided more direct evidence that miRNAs may contribute directly to causing cancer by forcing the over-expression of six miRNAs in mice that resulted in a significant increase in B cell lymphomas.

[0007] In humans, let-7 is thought to play a role in lung cancer development.
Let-7 expression is reduced in many lung cancer cell lines (Takamizawa et al., 2004) and in tumor samples relative to normal samples from lung cancer patients (Takamizawa et al., 2004; Johnson et al., 2005). Over-expression of let-7 inhibited growth of the lung cancer cell line, A549 (Takamizawa et al., 2004). Let-7 has been shown to reduce expression of RAS oncogenes in HepG2 cells (Johnson et al., 2005).
Together these data suggest that let-7 miRNAs may act as tumor suppressors in lung tissues.

[0008] Regulation of target genes by let-7 is thought to occur primarily by translation inhibition, but mRNA instability may also be a mechanism (Bagga et al., 2005, Reinhart et al., 2000). Besides RAS, the genes, gene pathways, and gene networks that are regulated by let-7 in cancerous cells remain largely unknown.
Currently, this represents a significant limitation for treatment of cancers in which let-7 may play a role.

[0009] In animals, most miRNAs are thought to regulate genes through imprecise base pairing within the 3' untranslated regions of their gene targets.
Bioinformatics analyses suggest that any given miRNA may bind to and alter the expression of up to several hundred different genes. Furthermore, a single gene may be regulated by several miRNAs. Thus, each miRNA may regulate a complex interaction among genes, gene pathways, and gene networks. Mis-regulation or alteration of these miRNA related regulatory pathways and networks are likely to contribute to the development of disorders, pathological conditions, and/or diseases such as cancer.
Although bioinformatics tools are helpful in predicting miRNA binding targets, all have limitations. Because of the imperfect complementarity with their target binding sites, it is difficult to precisely predict miRNA targets with bioinformatics tools alone.

[0010] Correcting gene expression errors by manipulating miRNA expression or by repairing miRNA mis-regulation represent promising methods to repair genetic disorders and cure diseases like cancer. A current, disabling limitation of this approach is that the details of the regulatory pathways and networks that are affected by any given miRNA remain largely unknown. As mentioned above, bioinformatics can provide only an imprecise estimate of the number and identity of miRNA
targets.
A need exists to identify the genes, genetic pathways, and genetic networks that are regulated by or that may regulate let-7 expression.

SUMMARY OF THE INVENTION

[0011] In particular aspects, a subject or patient may be selected for treatment based on expression and/or aberrant expression of one or more miRNA or mRNA.
In a further aspect, a subject or patient may be selected for treatment based on aberrations in one or more biologic or physiologic pathway(s), including aberrant expression of one or more gene associated with a pathway, or the aberrant expression of one or more protein encoded by one or more gene associated with a pathway.
In certain aspects, compositions of the invention are administered to a subject having, suspected of having, or at risk of developing a metabolic, an immunologic, an infectious, a cardiovascular, a digestive, an endocrine, an ocular, a genitourinary, a blood, a musculoskeletal, a nervous system, a congenital, a respiratory, a skin, or a cancerous disease or condition.

[0012] In still a further aspect, a subject or patient may be selected based on aberrations in miRNA expression, or biologic and/or physiologic pathway(s). A
subject may be assessed for sensitivity, resistance, and/or efficacy of a therapy or treatment regime based on the evaluation and/or analysis of miRNA or mRNA
expression or lack thereof. A subject may be evaluated for amenability to certain therapy prior to, during, or after administration of one or therapy to a subject or patient. Typically, evaluation or assessment may be done by analysis of miRNA
and/or mRNA, as well as combination of other assessment methods that include but are not limited to histology, immunohistochemistry, blood work, etc.

[0013] In some embodiments, an infectious disease or condition includes a bacterial, viral, parasite, or fungal infection. Many of these genes and pathways are associated with various cancers and other diseases. Cancerous conditions include, but are not limited to anaplastic large cell lymphoma, B-cell lymphoma, chronic lymphoblastic leukemia, multiple myeloma, testicular tumor, astrocytoma, acute myelogenous leukemia, breast carcinoma, bladder carcinoma, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, esophageal squamous cell carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lipoma, melanoma, mantle cell lymphoma, myxofibrosarcoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, lung carcinoma, non-small cell lung carcinoma, ovarian carcinoma, esophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, urothelial carcinoma wherein the modulation of one or more gene is sufficient for a therapeutic response. Typically a cancerous condition is an aberrant hyperproliferative condition associated with the uncontrolled growth or inability to undergo cell death, including apoptosis.

[0014] The present invention provides methods and compositions for identifying genes that are direct targets for let-7 regulation or are downstream targets of regulation following the hsa-let-7-mediated modification of upstream gene expression. Furthermore, the invention describes gene pathways and networks that are influenced by hsa-let-7 expression in biological samples. Many of these genes and pathways are associated with various cancers and other diseases. The altered expression or function of let-7 in cells would lead to changes in the expression of these key genes and contribute to the development of disease or other condition.
Introducing let-7 (for diseases where the miRNA is down-regulated) or a let-7 inhibitor (for diseases where the miRNA is up-regulated) into disease cells or tissues or subjects would result in a therapeutic response. The identities of key genes that are regulated directly or indirectly by let-7 and the disease with which they are associated are provided herein. In certain aspects a cell may be an epithelial, stromal, or mucosal cell. The cell can be, but is not limited to brain, a neuronal, a blood, an esophageal, a lung, a cardiovascular, a liver, a breast, a bone, a thyroid, a glandular, an adrenal, a pancreatic, a stomach, a intestinal, a kidney, a bladder, a prostate, a uterus, an ovarian, a testicular, a splenic, a skin, a smooth muscle, a cardiac muscle, or a striated muscle cell. In certain aspects, the cell, tissue, or target may not be defective in miRNA
expression yet may still respond therapeutically to expression or over expression of a miRNA. Let-7 could be used as a therapeutic target for any of these diseases.
In certain embodiments let-7 can be used to modulate the activity of let-7 in a subject, organ, tissue, or cell.

[0015] A cell, tissue, or subject may be a cancer cell, a cancerous tissue, harbor cancerous tissue, or be a subject or patient diagnosed or at risk of developing a disease or condition. In certain aspects a cancer cell is a neuronal, glial, lung, liver, brain, breast, bladder, blood, leukemic, colon, endometrial, stomach, skin, ovarian, fat, bone, cervical, esophageal, pancreatic, prostate, kidney, testicular or thyroid cell. In still a fiuther aspect cancer includes, but is not limited to anaplastic large cell lymphoma, B-cell lymphoma, chronic lymphoblastic leukemia, multiple myeloma, testicular tumor, astrocytoma, acute myelogenous leukemia, breast carcinoma, bladder carcinoma, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, esophageal squamous cell carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lipoma, melanoma, mantle cell lymphoma, myxofibrosarcoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, lung carcinoma, non-small cell lung carcinoma, ovarian carcinoma, esophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, urothelial carcinoma.

[0016] Embodiments of the invention include methods of modulating gene expression, or biologic or physiologic pathways in a cell, a tissue, or a subject comprising administering to the cell, tissue, or subject an amount of an isolated nucleic acid or mimetic thereof comprising a let-7 nucleic acid, mimetic, or inhibitor sequence in an amount sufficient to modulate the expression of a gene positively or negatively modulated by a let-7 miRNA family member. A "let-7 nucleic acid sequence" includes the full length precursor of a let-7 or complement thereof or processed (i.e., mature) sequence of let-7 and related sequences set forth herein, as well as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or more nucleotides of a precursor miRNA or its processed sequence, or complement thereof, including all ranges and integers there between. In certain embodiments, the let-7 nucleic acid sequence or let-7 inhibitor contains the fiill-length processed miRNA sequence or complement thereof and is referred to as the "let-full-length processed nucleic acid sequence" or "let-7 full-length processed inhibitor sequence." In still further aspects, the let-7 nucleic acid comprises at least one 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 nucleotide (including all ranges and integers there between) segment or complementary segment of a let-7 that is at least 75, 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NO:1 to SEQ ID NO: 11. The general term let-7 includes all members of the let-7 family that share at least part of a mature let-7 sequence.

[0017] In certain aspects, a let-7 nucleic acid, or a segment or a mimetic thereof, will comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or more nucleotides of the precursor miRNA or its processed sequence, including all ranges and integers there between. In certain embodiments, the let-7 nucleic acid sequence contains the full-length processed miRNA sequence and is referred to as the "let-7 full-length processed nucleic acid sequence." In still further aspects, a let-7 comprises at least one 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 nucleotide (including all ranges and integers there between) segment of let-7 that is at least 75, 80, 85, 90, 95, 98, 99 or 100%
identical to SEQ ID NOs provided herein.

[0018] In specific embodiments, a let-7 or let-7 inhibitor containing nucleic acid is a hsa- let-7 or hsa- let-7 inhibitor or a variation thereof. In a further aspect, a let-7 nucleic acid or let-7 inhibitor can be administered with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more miRNAs or miRNA inhibitors. miRNAs or their complements can be administered concurrently, sequentially, or in an ordered progression. In certain aspects, a let-7 or let-7 inhibitor can be administered in combination with one or more of miR-15, miR-16, miR-20, miR-21, miR-26a, miR-34a, miR-124, miR-126, miR-143, miR-147, miR-188, miR-200, miR-215, miR-216, miR-292-3p, and/or miR-331.
All or combinations of miRNAs or inhibitors thereof may be administered in a single formulation. Administration may be before, during or affter a second therapy.

[0019] let-7 nucleic acids or complement thereof may also include various heterologous nucleic acid sequences, i.e., those sequences not typically found operatively coupled with let-7 in nature, such as promoters, enhancers, and the like.
The let-7 nucleic acid is a recombinant nucleic acid, and can be a ribonucleic acid or a deoxyribonucleic acid. The recombinant nucleic acid may comprise a let-7 or let-7 inhibitor expression cassette, i.e., a nucleic acid segment that expresses a nucleic acid when introduce into an environment containing components for nucleic acid synthesis. In a further aspect, the expression cassette is comprised in a viral vector, or plasmid DNA vector or otber therapeutic nucleic acid vector or delivery vehicle, including liposomes and the like. In a particular aspect, the let-7 nucleic acid is a synthetic nucleic acid. Moreover, nucleic acids of the invention may be fuily or partially synthetic. In certain aspects, viral vectors can be administered at 1x102, 1x103> 1x104 1x105> ix106, 1x107, 1x10I, 1x109, 1x1010, lxl011, 1x1012, 1x1013 , 1x1014 pfu or viral particle (vp).

[0020] In a particular aspect, the let-7 nucleic acid or let-7 inhibitor is a synthetic nucleic acid. Moreover, nucleic acids of the invention may be fully or partially synthetic. In still further aspects, a nucleic acid of the invention or a DNA
encoding such a nucleic acid of the invention can be administered at 0.001, 0.01, 0.1, 1, 10, 20, 30, 40, 50, 100, 200, 400, 600, 800, 1000, 2000, to 4000 g or mg, including all values and ranges there between. In yet a further aspect, nucleic acids of the invention, including synthetic nucleic acid, can be administered at 0.001, 0.01, 0.1, 1, 10, 20, 30, 40, 50, 100, to 200 g or mg per kilogram (kg) of body weight.
Each of the amounts described herein may be administered over a period of time, including 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, minutes, hours, days, weeks, months or years, including all values and ranges there between.

[0021] In certain embodiments, administration of the composition(s) can be enteral or parenteral. In certain aspects, enteral administration is oral. In further aspects, parenteral administration is intralesional, intravascular, intracranial, intrapleural, intratumoral, intraperitoneal, intramuscular, intralymphatic, intraglandular, subcutaneous, topical, intrabronchial, intratracheal, intranasal, inhaled, or instilled. Compositions of the invention may be administered regionally or locally and not necessarily directly into a lesion.

[0022] In certain aspects, the gene or genes modulated comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200 or more genes or combinations of genes identified in Table 2, 3, 4, 5, 6, 7, and 8. In certain aspects the expression of a gene is down-regulated or up-regulated. In a particular aspect the gene modulated comprises or is selected from (and may even exclude) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26.
27, 28, or all of ATRX, AURKA/STK6, AURKB/STK12, BRCAl, BRCA2, BUB1, BUB1B, BZRP, CCNA2, CCNB1, CCNE2, CCNG2, CDC2, CDC20, CDC23, CDC25A, CDC6, CDCA7, CDK2, CDK6, CDKN2B, CDT1, CEBPD, CKS1B, CSFl, EIF4E, EPHB2, ERBB3, FASN, FGFBPI, FGFR4, FH, GMNN, IGFBP, IL8, ITGA6, JUN, JUNB, LHFP, MCAM, MET, MVP, MXI1, MYBL1, MYBL2, NRAS, P8, PDCD4, PLKl, PRKCA, RASSF2, SIVA, SKP2, SMAD4, TACC3, TFDPi, TGFBR3, TNFSFIO, and/or VIM, in various combinations and permutations. In certain embodiments a gene modulated or selected to be modulated is from Table 2. In further embodiments a gene modulated or selected to be modulated is from Table 3.
In still further embodiments a gene modulated or selected to be modulated is from Table 4. In certain embodiments a gene modulated or selected to be modulated is from 5. In further embodiments a gene modulated or selected to be modulated is from 6. In still further embodiments a gene modulated or selected to be modulated is from 7. In certain embodiments a gene modulated or selected to be modulated is from 8.
Embodiments of the invention may also include obtaining or assessing a gene expression profile or miRNA profile of a target cell prior to selecting the mode of treatment, e.g., administration of a let-7 nucleic acid, inhibitor of let-7, or mimetics thereof. The database content related to all nucleic acids and genes designated by an accession number or a database submission are incorporated herein by reference as of the filing date of this application. . In certain aspects of the invention one or more miRNA or miRNA inhibitor may modulate a single gene. In a further aspect, one or more genes in one or more genetic, cellular, or physiologic pathways can be modulated by one or more miRNAs or complements thereof, including let-7 nucleic acids and let-7 inhibitors in combination with other miRNAs.

[0023] In still further aspects, the let-7 nucleic acid comprises at least one of hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-let-7g, hsa-let-7i, or a segment thereof. A cell, tissue, or subject may be a cancer cell, a cancerous tissue or harbor cancerous tissue, or a cancer patient. In a particular aspect the cancer is blood, leukemic, colon, endometrial, stomach, skin, ovarian, esophageal, pancreatic, prostate, salivary gland, small intestine, thyroid, lung or liver cancer.

[0024] Let-7 nucleic acids may also include various heterologous nucleic acid sequences, i.e., those sequences not typically found operatively coupled with let-7 in nature, such as promoters, enhancers, and the like. The let-7 nucleic acid is a recombinant nucleic acid, and can be a ribonucleic acid or a deoxyribonucleic acid.
The recombinant nucleic acid may comprise a let-7 expression cassette. In a further aspect, the expression cassette is comprised in a viral, or plasmid DNA vector or other therapeutic nucleic acid vector or delivery vehicle, including liposomes and the like.
In a particular aspect, the let-7 nucleic acid is a synthetic nucleic acid.

[0025] A further embodiment of the invention is directed to methods of modulating a cellular pathway comprising administering to the cell an amount of an isolated nucleic acid comprising a let-7 nucleic acid sequence in an amount sufficient to modulate the expression, function, status, or state of a cellular pathway described in Table 13 or the pathways known to include one or more genes identified herein as modulated by let-7. Modulation of a cellular pathway includes, but is not limited to modulating the expression of one or more gene identified in Table 2, 3, 4, 5, 6, 7, and 8. Modulation of a gene can include inhibiting the function of an endogenous miRNA
or providing a functional miRNA to a cell, tissue, or subject. Modulation refers to the expression levels or activities of a gene or its related gene product or protein, e.g., the mRNA levels may be modulated or the translation of an mRNA may be modulated, etc. Modulation may increase or up regulate a gene or gene product or it may decrease or down regulate a gene or gene product.

[0026] Still a further embodiment includes methods of treating a patient with a pathological condition comprising one or more of step (a) administering to the patient an amount of an isolated nucleic acid comprising a let-7 nucleic acid sequence in an amount sufficient to modulate the expression of a cellular pathway; and (b) administering a second therapy, wherein the modulation of the cellular pathway sensitizes the patient to the second therapy. A cellular pathway may include, but is not limited to one or more pathway described in the Tables herein or a pathway that is known to include one or more genes listed in the Tables. A second therapy can include administration of a second miRNA or therapeutic nucleic acid, or may include various standard therapies, such as chemotherapy, radiation therapy, drug therapy, immunotherapy, and the like. Embodiments of the invention may also include the determination or assessment of a gene expression profile for the selection of an appropriate therapy.

[0027] Embodiments of the invention include methods of treating a subject with a pathological condition comprising one or more of the steps of (a) determining an expression profile of one or more genes selected from Table 2, 3, 4, 5, 6, 7, and 8; (b) assessing the sensitivity of the subject to therapy based on the expression profile; (c) selecting a therapy based on the assessed sensitivity; and (d) treating the subject using selected therapy. Typically, the pathological condition will have as a component, indicator, or result the mis-regulation of one or more gene of Table 2, 3, 4, 5, 6, 7, and 8.

[0028] Further embodiments include the identification and assessment of an expression profile indicative of let-7 status in a cell or tissue comprising expression assessment of one or more gene from Table 2, 3, 4, 5, 6, 7, and 8, or any combination thereof.

[0029] The term "miRNA" is used according to its ordinary and plain meaning and refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation. See, e.g., Carrington et al., 2003, which is hereby incorporated by reference. The term can be used to refer to the single-stranded RNA
molecule processed from a precursor or in certain instances the precursor itself.

[0030] In some embodiments, it may be useful to know whether a cell expresses a particular miRNA endogenously or whether such expression is affected under particular conditions or when it is in a particular disease state. Thus, in some embodiments of the invention, methods include assaying a cell or a sample containing a cell for the presence of one or more marker gene or mRNA or other analyte indicative of the expression level of a gene of interest. Consequently, in some embodiments, methods include a step of generating an RNA profile for a sample.
The term "RNA profile" or "gene expression profile" refers to a set of data regarding the expression pattern for one or more gene or genetic marker in the sample (e.g., a plurality of nucleic acid probes that identify one or more markers from Table 2); it is contemplated that the nucleic acid profile can be obtained using a set of RNAs, using for example nucleic acid amplification or hybridization techniques well know to one of ordinary skill in the art. The difference in the expression profile in the sample from the patient and a reference expression profile, such as an expression profile from a normal or non-pathologic sample, is indicative of a pathologic, disease, or cancerous condition. A nucleic acid or probe set comprising or identifying a segment of a corresponding mRNA can include all or part of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 ,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 100, 200, 500, or more nucleotides, including any integer or range derivable there between, of a gene or genetic marker, or a nucleic acid, mRNA
or a probe representative thereof that is listed in Table 2, 3, 4, 5, 6, 7, and 8 or identified by the methods described herein.

[00311 Certain embodiments of the invention are directed to compositions and methods for assessing, prognosing, or treating a pathological condition in a patient comprising measuring or determining an expression profile of one or more marker(s) in a sample from the patient, wherein a difference in the expression profile in the sample from the patient and an expression profile of a normal sample or reference expression profile is indicative of pathological condition and particularly cancer (e.g., In certain aspects of the invention, the cellular pathway, gene, or genetic marker is or is representative of one or more pathway or marker described in Table 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and/or 13, including any combination thereof.

[0032] Aspects of the invention include diagnosing, assessing, or treating a pathologic condition or preventing a pathologic condition from manifesting.
For example, the methods can be used to screen for a pathological condition;
assess prognosis of a pathological condition; stage a pathological condition; assess response of a pathological condition to therapy; or to modulate the expression of a gene, genes, or related pathway as a first therapy or to render a subject sensitive or more responsive to a second therapy. In particular aspects, assessing the pathological condition of the patient can be assessing prognosis of the patient. Prognosis may include, but is not limited to an estimation of the time or expected time of survival, assessment of response to a therapy, and the like. In certain aspects, the altered expression of one or more gene or marker is prognostic for a patient having a pathologic condition, wherein the marker is one or more of Table 2, 3, 4, 5, 6, 7, 8, 12 and/or 13, including any combination thereof.

[0033] A further embodiment of the invention is directed to methods of modulating a cellular pathway comprising administering to the cell an amount of an isolated nucleic acid comprising a let-7 nucleic acid sequence or a let-7 inhibitor. A
cell, tissue, or subject may be a cancer cell, a cancerous tissue or harbor cancerous tissue, or a cancer patient. The database content related to all nucleic acids and genes designated by an accession number or a database submission are incorporated herein by reference as of the filing date of this application.

[0034] A further embodiment of the invention is directed to methods of modulating a cellular pathway comprising administering to the cell an amount of an isolated nucleic acid comprising a let-7 nucleic acid sequence in an amount sufficient to modulate the expression, function, status, or state of a cellular pathway, in particular those pathways described in Table 2, 3, 4, 5, 6, 7, and 8 or the pathways known to include one or more genes from Table 2, 3, 4, 5, 6, 7, and 8.
Modulation of a cellular pathway includes, but is not limited to modulating the expression of one or more gene(s). Modulation of a gene can include inhibiting the function of an endogenous miRNA or providing a functional miRNA to a cell, tissue, or subject.
Modulation refers to the expression levels or activities of a gene or its related gene product (e.g., mRNA) or protein, e.g., the mRNA levels may be modulated or the translation of an mRNA may be modulated. Modulation may increase or up regulate a gene or gene product or it may decrease or down regulate a gene or gene product (e.g., protein levels or activity).

[0035] Still a further embodiment includes methods of administering an miRNA
or mimic thereof, and/or treating a subject or patient having, suspected of having, or at risk of developing a pathological condition comprising one or more of step (a) administering to a patient or subject an amount of an isolated nucleic acid comprising a let-7 nucleic acid sequence or a let-7 inhibitor in an amount sufficient to modulate expression of a cellular pathway; and (b) administering a second therapy, wherein the modulation of the cellular pathway sensitizes the patient or subject, or increases the efficacy of a second therapy. An increase in efficacy can include a reduction in toxicity, a reduced dosage or duration of the second therapy, or an additive or synergistic effect. A cellular pathway may include, but is not limited to one or more ' pathway described in Table 2, 3, 4, 5, 6, 7, and 8 below or a pathway that is know to include one or more genes of Table 2, 3, 4, 5, 6, 7, and 8. The second therapy may be administered before, during, and/or after the isolated nucleic acid or miRNA
or inhibitor is administered [0036] A second therapy can include administration of a second miRNA or therapeutic nucleic acid such as a siRNA or antisense oligonucleotide, or may include various standard therapies, such as pharmaceuticals, chemotherapy, radiation therapy, drug therapy, immunotherapy, and the like. Embodiments of the invention may also include the determination or assessment of gene expression or gene expression profile for the selection of an appropriate therapy. In a particular aspect, a second therapy is chemotherapy. A chemotherapy can include, but is not limited to paclitaxel, cisplatin, carboplatin, doxorubicin, oxaliplatin, larotaxel, taxol, lapatinib, docetaxel, methotrexate, capecitabine, vinorelbine, cyclophosphamide, gemcitabine, amrubicin, cytarabine, etoposide, camptothecin, dexamethasone, dasatinib, tipifamib, bevacizumab, sirolimus, temsirolimus, everolimus, lonafarnib, cetuximab, erlotinib, gefitinib, imatinib mesylate, rituximab, trastuzumab, nocodazole, sorafenib, sunitinib, bortezomib, alemtuzumab, gemtuzumab, tositumomab or ibritumomab.

[0037] Embodiments of the invention include methods of treating a subject with a disease or condition comprising one or more of the steps of (a) determining an expression profile of one or more genes selected from Table 2, 3, 4, 5, 6, 7, and 8; (b) assessing the sensitivity of the subject to therapy based on the expression profile; (c) selecting a therapy based on the assessed sensitivity; and (d) treating the subject using a selected therapy. Typically, the disease or condition will have as a component, indicator, or resulting mis-regulation of one or more gene of Table 2, 3, 4, 5, 6, 7, and 8.

[0038] In certain aspects, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more miRNA may be used in sequence or in combination. For instance, any combination of let-7 or a let-7 inhibitor with another miRNA. Further embodiments include the identification and assessment of an expression profile indicative of let-7 status in a cell or tissue comprising expression assessment of one or more gene from Table 2, 3, 4, 5, 6, 7, and 8, or any combination thereof.

[0039] The term "miRNA" is used according to its ordinary and plain meaning and refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation. See, e.g., Carrington et al., 2003, which is hereby incorporated by reference. The term can be used to refer to the single-stranded RNA
molecule processed from a precursor or in certain instances the precursor itself.

[0040] In some embodiments, it may be useful to know whether a cell expresses a particular miRNA endogenously or whether such expression is affected under particular conditions or when it is in a particular disease state. Thus, in some embodiments of the invention, methods include assaying a cell or a sample containing a cell for the presence of one or more marker gene or mRNA or other analyte indicative of the expression level of a gene of interest. Consequently, in some embodiments, methods include a step of generating an RNA profile for a sample.
The term "RNA profile" or "gene expression profile" refers to a set of data regarding the expression pattern for one or more gene or genetic marker or miRNA in the sample (e.g., a plurality of nucleic acid probes that identify one or more markers from Table 2, 3, 4, 5, 6, 7, and 8); it is contemplated that the nucleic acid profile can be obtained using a set of RNAs, using for example nucleic acid amplification or hybridization techniques well know to one of ordinary skill in the art. The difference in the expression profile in the sample from the patient and a reference expression profile, such as an expression profile of one or more genes or miRNAs, are indicative of which miRNAs to be administered.

[0041] In certain aspects, let-7 or let-7 inhibitor and miR-10 or miR-10 inhibitor are administered to patients with astrocytoma, acute lymphoblastic leukemia, acute myeloid leukemia, breast carcinoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, chronic myeloid leukemia, colorectal carcinoma, endometrial carcinoma, fibrosarcoma, glioma, glioblastoma, glioblastoma multiforme, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, leukeinia, lung carcinoma, leiomyoma, melanoma, multiple myeloma, mesothelioma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, retinoblastoma, renal cell carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, T-cell leukemia.

[0042] In yet another aspect, let-7 or let-7 inhibitor and miR-15 or miR-15 inhibitor can be administered to patients with astrocytoma, acute myeloid leukemia, breast carcinoma, bladder carcinoma, cervical carcinoma, carcinoma of the head and neck, chronic myeloid leukemia, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, lung carcinoma, larynx carcinoma, melanoma, medulloblastoma, myxofibrosarcoma, myeloid leukemia, multiple myeloma, neuroblastorna, non-Hodgkin lymphoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, thyroid carcinoma.

[0043] Further aspects include administering let-7 or let-7 inhibitor and miR-16 or miR-16 inhibitor to patients with astrocytoma, breast carcinoma, bladder carcinoma, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, melanoma, medulloblastoma, myxofibrosarcoma, myeloid leukemia, multiple myeloma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, thyroid carcinoma.

[0044] In still further aspects, let-7 or let-7 inhibitor and miR-20 or miR-20 inhibitor are administered to patients with astrocytoma, acute myeloid leukemia, breast carcinoma, bladder carcinoma, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lipoma, melanoma, myxofibrosarcoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, urothelial carcinoma.

[0045] In certain aspects, let-7 or let-7 inhibitor and miR-21 or miR-21 inhibitor are administered to patients with astrocytoma, acute lymphoblastic leukemia, acute myeloid leukemia, breast carcinoma, Burkitt's lymphoma, bladder carcinoma, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, melanoma, myeloid leukemia, neuroblastoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck.

[0046] Aspects of the invention include methods where let-7 or let-7 inhibitor and miR-26 or miR-26 inhibitor are administered to patients with acute lymphoblastic leukemia, acute myeloid leukemia, angiosarcoma, breast carcinoma, Burkitt's lymphoma, bladder carcinoma, cervical carcinoma, carcinoma of the head and neck, chronic lymphoblastic leukemia, chronic myeloid leukemia, colorectal carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Kaposi's sarcoma, leukemia, lung carcinoma, leiomyosarcoma, larynx carcinoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, rhabdomyosarcoma.

[0047] In still further aspects, let-7 or let-7 inhibitor and miR-34 or miR-34 inhibitor are administered to patients with astrocytoma, acute lymphoblastic leukemia, acute myeloid leukemia, angiosarcoma, breast carcinoma, bladder carcinoma, cervical carcinoma, carcinoma of the head and neck, chronic lymphoblastic leukemia, chronic myeloid leukemia, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, gastrinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, Kaposi's sarcoma, leukemia, lung carcinoma, leiomyosarcoma, melanoma, medulloblastoma, myeloid leukemia, multiple myeloma, mesothelioma, non-Hodgkin lymphoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, oropharyngeal carcinoma, osteosarcoma, pancreatic carcinoma, papillary carcinoma, prostate carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, salivary gland tumor, sporadic papillary renal carcinoma, thyroid carcinoma, urothelial carcinoma.

[0048] In still a further aspect, let-7 or let-7 inhibitor and miR-124 or miR-inhibitor are administered to patients with astrocytoma, acute lymphoblastic leukemia, acute myeloid leukemia, angiosarcoma, breast carcinoma, Burkitt's lymphoma, bladder carcinoma, cervical carcinoma, carcinoma of the head and neck, chronic lymphoblastic leukemia, chronic myeloid leukemia, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, glioblastoma multiforme, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, Kaposi's sarcoma, leukemia, lung carcinoma, lipoma, leiomyosarcoma, melanoma, medulloblastoma, myxofibrosarcoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, oropharyngeal carcinoma, osteosarcoma, pancreatic carcinoma, papillary carcinoma, prostate carcinoma, retinoblastoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, salivary gland tumor, thyroid carcinoma, urothelial carcinoma.

[0049] In yet further aspects, let-7 or let-7 inhibitor and miR-126 or miR-126 inhibitor are administered to patients with astrocytoma, acute myeloid leukemia, breast carcinoma, Burkitt's lymphoma, bladder carcinoma, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, gastrinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lung carcinoma, melanoma, myeloid leukemia, mesothelioma, non-Hodgkin lymphoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, oropharyngeal carcinoma, osteosarcoma, pancreatic carcinoma, papillary carcinoma, prostate carcinoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, sporadic papillary renal carcinoma, thyroid carcinoma.

[0050] In yet further aspects, let-7 or let-7 inhibitor and miR-143 or miR-143 inhibitor are administered to patients with astrocytoma, acute lymphoblastic leukemia, acute myeloid leukemia, breast carcinoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, chronic myeloid leukemia, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lung carcinoma, melanoma, medulloblastoma, multiple myeloma, non-Hodgkin lymphoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma.

[0051] In a further aspect, let-7 or let-7 inhibitor and miR-147 or miR-147 inhibitor are administered to patients with astrocytoma, breast carcinoma, bladder carcinoma, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lipoma, melanoma, myxofibrosarcoma, multiple myeloma, non-Hodgkin lymphoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma.

[0052] In still a further aspect, let-7 or let-7 inhibitor and miR-188 or miR-inhibitor are administered to patients with astrocytoma, acute myeloid leukemia, breast carcinoma, Burkitt's lymphoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, leukemia, lung carcinoma, melanoma, multiple myeloma, non-Hodgkin lymphoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma.

[0053] In a fizrther aspect, let-7 or let-7 inhibitor and miR-200 or miR-200 inhibitor are administered to patients with breast carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, leukemia, lung carcinoma, lipoma, multiple myeloma, mesothelioma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, thyroid carcinoma.

[0054] In yet another aspect, let-7 or let-7 inhibitor and miR-215 or miR-215 inhibitor are administered to patients with astrocytoma, acute lymphoblastic leukemia, acute myeloid leukemia, angiosarcoma, breast carcinoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, chronic myeloid leukemia, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, gastrinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, Kaposi's sarcoma, leukemia, lung carcinoma, lipoma, leiomyosarcoma, melanoma, myxofibrosarcoma, myeloid leukemia, multiple myeloma, mesothelioma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, retinoblastoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, salivary gland tumor, thyroid carcinoma, urothelial carcinoma.

[0055] In yet a further aspect, let-7 or let-7 inhibitor and miR-216 or miR-inhibitor are administered to patients with astrocytoma, breast carcinoma, cervical carcinoma, carcinoma of the head and neck, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lung carcinoma, myeloid leukemia, non-Hodgkin lymphoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, osteosarcoma, prostate carcinoma, squamous cell carcinoma of the head and neck.

[0056] In other aspects, let-7 or let-7 inhibitor and miR-292-3p or miR-292-3p inhibitor are administered to patients with astrocytoma, acute lymphoblastic leukemia, acute myeloid leukemia, angiosarcoma, breast carcinoma, bladder carcinoma, cervical carcinoma, chronic myeloid leukemia, colorectal carcinoma, endometrial carcinoma, fibrosarcoma, glioma, glioblastoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Kaposi's sarcoma, leukemia, lung carcinoma, lipoma, leiomyosarcoma, melanoma, myxofibrosarcoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, urothelial carcinoma.

[0057] In certain aspects, let-7 or let-7 inhibitor and miR-331 or miR-331 inhibitor are administered to patients with astrocytoma, acute lymphoblastic leukemia, acute myeloid leukemia, angiosarcoma, breast carcinoma, bladder carcinoma, cervical carcinoma, carcinoma of the head and neck, chronic lymphoblastic leukemia, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, gastrinoma, hepatocellular carcinoma, Kaposi's sarcoma, leukemia, lung carcinoma, leiomyosarcoma, larynx carcinoma, melanoma, myxofibrosarcoma, myeloid leukemia, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, ovarian carcinoma, oesophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, sporadic papillary renal carcinoma, thyroid carcinoma.

[0058] It is contemplated that when let-7 or a let-7 inhibitor is given in combination with one or more other miRNA molecules, the two different miRNAs or inhibitors may be given at the same time or sequentially. In some embodiments, therapy proceeds with one miRNA or inhibitor and that therapy is followed up with therapy with the other miRNA or inhibitor 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or any such combination later.

[0059] Further embodiments include the identification and assessment of an expression profile indicative of let-7 status in a cell or tissue comprising expression assessment of one or more gene from Table 2, 3, 4, 5, 6, 7, and 8, or any combination thereof.

[0060] The term "miRNA" is used according to its ordinary and plain meaning and refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation. See, e.g., Carrington et al., 2003, which is hereby incorporated by reference. The term can be used to refer to the single-stranded RNA
molecule processed from a precursor or in certain instances the precursor itself or a mimetic thereof.

[0061] In some embodiments, it may be useful to know whether a cell expresses a particular miRNA endogenously or whether such expression is affected under particular conditions or when it is in a particular disease state. Thus, in some embodiments of the invention, methods include assaying a cell or a sample containing a cell for the presence of one or more miRNA marker gene or mRNA or other analyte indicative of the expression level of a gene of interest. Consequently, in some embodiments, methods include a step of generating an RNA profile for a sample.
The term "RNA profile" or "gene expression profile" refers to a set of data regarding the expression pattem for one or more gene or genetic marker in the sample (e.g., a plurality of nucleic acid probes that identify one or more markers or genes from Table 2, 3, 4, 5, 6, 7, and 8); it is contemplated that the nucleic acid profile can be obtained using a set of RNAs, using for example nucleic acid amplification or hybridization techniques well know to one of ordinary skill in the art. The difference in the expression profile in the sample from a patient and a reference expression profile, such as an expression profile from a normal or non-pathologic sample, or a digitized reference, is indicative of a pathologic, disease, or cancerous condition. In certain aspects the expression profile is an indicator of a propensity to or probability of (i.e., risk factor for a disease or condition) developing such a condition(s). Such a risk or propensity may indicate a treatment, increased monitoring, prophylactic measures, and the like. A nucleic acid or probe set may comprise or identify a segment of a corresponding mRNA and may include all or part of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 ,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 100, 200, 500, or more segments, including any integer or range derivable there between, of a gene or genetic marker, or a nucleic acid, mRNA
or a probe representative thereof that is listed in Table 2, 3, 4, 5, 6, 7, and 8 or identified by the methods described herein.

[0062] Certain embodiments of the invention are directed to compositions and methods for assessing, prognosing, or treating a pathological condition in a patient comprising measuring or determining an expression profile of one or more miRNA
or marker(s) in a sample from the patient, wherein a difference in the expression profile in the sample from the patient and an expression profile of a normal sample or reference expression profile is indicative of pathological condition and particularly cancer (e.g., In certain aspects of the invention, the miRNAs, cellular pathway, gene, or genetic marker is or is representative of one or more pathway or marker described in Table 2, 3, 4, 5, 6, 7, and 8, including any combination thereof.

[0063] Aspects of the invention include diagnosing, assessing, or treating a pathologic condition or preventing a pathologic condition from manifesting.
For example, the methods can be used to screen for a pathological condition;
assess prognosis of a pathological condition; stage a pathological condition; assess response of a pathological condition to therapy; or to modulate the expression of a gene, genes, or related pathway as a first therapy or to render a subject sensitive or more responsive to a second therapy. In particular aspects, assessing the pathological condition of the patient can be assessing prognosis of the patient. Prognosis may include, but is not limited to an estimation of the time or expected time of survival, assessment of response to a therapy, and the like. In certain aspects, the altered expression of one or more gene or marker is prognostic for a patient having a pathologic condition, wherein the marker is one or more of Table 2, 3, 4, 5, 6, 7, and 8, including any combination thereof.

[0064] Certain embodiments of the invention include determining expression of one or more marker, gene, or nucleic acid representative thereof, by using an amplification assay, a hybridization assay, or protein assay, a variety of which are well known to one of ordinary skill in the art. In certain aspects, an amplification assay can be a quantitative amplification assay, such as quantitative RT-PCR
or the like. In still further aspects, a hybridization assay can include array hybridization assays or solution hybridization assays. The nucleic acids from a sample may be labeled from the sample and/or hybridizing the labeled nucleic acid to one or more nucleic acid probes. Nucleic acids, mRNA, and/or nucleic acid probes may be coupled to a support. Such supports are well known to those of ordinary skill in the art and include, but are not limited to glass, plastic, metal, or latex. In particular aspects of the invention, the support can be planar or in the form of a bead or other geometric shapes or configurations known in the art. Proteins are typically assayed by immunoblotting, chromatography, or mass spectrometry or other methods known to those of ordinary skill in the art.

[0065] The present invention also concerns kits containing compositions of the invention or compositions to implement methods of the invention. In some embodiments, kits can be used to evaluate one or more marker molecules, and/or express one or more miRNA. In certain embodiments, a kit contains, contains at least or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 100, 150, 200 or more probes, recombinant nucleic acid, or synthetic nucleic acid molecules related to the markers to be assessed or an miRNA to be expressed or modulated, and may include any range or combination derivable therein. Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means. Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more. Kits for using probes, synthetic nucleic acids, recombinant nucleic acids, or non-synthetic nucleic acids of the invention for therapeutic, prognostic, or diagnostic applications are included as part of the invention. Specifically contemplated are any such molecules corresponding to any miRNA reported to influence biological activity or expression of one or more marker gene or gene pathway described herein. In certain aspects, negative and/or positive controls are included in some kit embodiments. The control molecules can be used to verify transfection efficiency and/or control for transfection-induced changes in cells.

[0066] Certain embodiments are directed to a kit for assessment of a pathological condition or the risk of developing a pathological condition in a patient by nucleic acid profiling of a sample comprising, in suitable container means, two or more nucleic acid hybridization or amplification reagents. The kit can comprise reagents for labeling nucleic acids in a sample and/or nucleic acid hybridization reagents. The hybridization reagents typically comprise hybridization probes. Amplification reagents include, but are not limited to ainplification primers, reagents, and enzymes.
[0067] In some embodiments of the invention, an expression profile is generated by steps that include: (a) labeling nucleic acid in the sample; (b) hybridizing the nucleic acid to a number of probes, or amplifying a number of nucleic acids, and (c) determining and/or quantitating nucleic acid hybridization to the probes or detecting and quantitating amplification products, wherein an expression profile is generated.
See U.S. Provisional Patent Application 60/575,743 and the U.S. Provisional Patent Application 60/649,584, and U.S. Patent Application Serial No. 11/141,707 and U.S.
Patent Application Serial No. 11/273,640, all of which are hereby incorporated by reference.

[0068] Methods of the invention involve diagnosing and/or assessing the prognosis of a patient based on a miRNA and/or a marker nucleic acid expression profile. In certain embodiments, the elevation or reduction in the level of expression of a particular gene or genetic pathway or set of nucleic acids in a cell is correlated with a disease state or pathological condition compared to the expression level of the same in a normal or non-pathologic cell or tissue sample. This correlation allows for diagnostic and/or prognostic methods to be carried out when the expression level of one or more nucleic acid is measured in a biological sample being assessed and then compared to the expression level of a normal or non-pathologic cell or tissue sample.
It is specifically contemplated that expression profiles for patients, particularly those suspected of having or having a propensity for a particular disease or condition such as cancer, can be generated by evaluating any of or sets of the miRNAs and/or nucleic acids discussed in this application. The expression profile that is generated from the patient will be one that provides information regarding the particular disease or condition. In many embodiments, the profile is generated using nucleic acid hybridization or amplification, (e.g., array hybridization or RT-PCR). In certain aspects, an expression profile can be used in conjunction with other diagnostic and/or prognostic tests, such as histology, protein profiles in the serum and/or cytogenetic assessment.

[0069] The methods can further comprise one or more of the steps including:
(a) obtaining a sample from the patient, (b) isolating nucleic acids from the sample, (c) labeling the nucleic acids isolated from the sample, and (d) hybridizing the labeled nucleic acids to one or more probes. Nucleic acids of the invention include one or more nucleic acid comprising at least one segment having a sequence or complementary sequence of to a nucleic acid representative of one or more of genes or markers in Table 2, 3, 4, 5, 6, 7, 8, and/or 12.

[0070] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined. It is specifically contemplated that any methods and compositions discussed herein with respect to miRNA molecules, miRNA, genes and nucleic acids representative of genes may be implemented with respect to synthetic nucleic acids. In some embodiments the synthetic nucleic acid is exposed to the proper conditions to allow it to become a processed or mature nucleic acid, such as a miRNA under physiological circumstances. The claims originally filed are contemplated to cover claims that are multiply dependent on any filed claim or combination of filed claims.

[0071] Also, any embodiment of the invention involving specific genes (including representative fragments there of), mRNA, or miRNAs by name is contemplated also to cover embodiments involving miRNAs whose sequences are at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the mature sequence of the specified miRNA.

[0072] It will be further understood that shorthand notations are employed such that a generic description of a gene or marker thereof or of a miRNA refers to any of its gene family members (distinguished by a number) or representative fragments thereof, unless otherwise indicated. It is understood by those of skill in the art that a "gene family" refers to a group of genes having the same coding sequence or miRNA
coding sequence. Typically, miRNA members of a gene family are identified by a number following the initial designation. For example, miR-16-1 and miR-16-2 are members of the miR-16 gene family and "mir-7" refers to rniR-7-1, miR-7-2 and miR-7-3. Moreover, unless otherwise indicated, a shorthand notation refers to related miRNAs (distinguished by a letter). Thus, "let-7," for example, refers to let-7a, let-7b, let-7c, let-7d, let-7e, I and the like. Exceptions to this shorthand notation will be otherwise identified.

Table 1. Listing of miRNA for diagnosis and thera y.
miRNA Probe segment miR Base Information Precursor sequence hsa-let-7a-I SEQ ID NO:1 >hsa-let-7a MIMAT0000062 SEQ ID NO: 12 hsa-let-7a-2 SEQ ID NO:2 SEQ ID NO:13 hsa-let-7a-3 SEQ ID NO:3 SEQ ID NO:14 hsa-let-7b SEQ ID NO:4 >hsa-let-7b MIMAT0000063 SEQ ID NO: 15 hsa-let-7c SEQ ID NO:5 >hsa-Iet-7c MIMAT0000064 SEQ ID NO: 16 hsa-let-7d SEQ ID NO:6 >hsa-let-7d MIMAT0000065 SEQ ID NO:17 hsa-let-7e SEQ ID NO:7 >hsa-let-7e MIMAT0000066 SEQ ID NO:18 hsa-let-7f-I SEQ ID NO:8 >hsa-Iet-7f MIMAT0000067 SEQ ID NO:19 hsa-let-7f-2 SEQ ID NO:9 SEQ ID NO:20 hsa-let-7g SEQ ID NO:10 >hsa-let-7g MIMAT0000414 SEQ ID NO:21 hsa-let-7i SEQ ID NO:1 I >hsa-let-7i MIMAT0000415 SEQ ID NO:22 [0073] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. The embodiments in the Example and Detailed Description section are understood to be embodiments of the invention that are applicable to all aspects of the invention.

[0074] The terms "inhibiting," "reducing," or "prevention," or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.

[0075] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0076] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0077] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

[0078] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0079] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

[0080] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0081] FIG. 1. Percent (%) proliferation of hsa-let-7 treated cells relative to cells treated with negative control miRNA (100%). Abbreviations: let-7b, hsa-let-7b;
let-7c, hsa-let-7c; let-7g, hsa-let-7g; siEg5, siRNA against the motor protein kinesin 11 (Eg5); Etopo, etoposide; NC, negative control miRNA. Standard deviations are indicated in the graph.

[0082] FIG. 2. Dose dependent inhibition of various cell lines by hsa-let-7 using Alamar Blue proliferation assays. Cell proliferation is reported as %
proliferation relative to % proliferation of mock-transfected cells (0 pM = 100%
proliferation).
Standard deviations are indicated in the graphs. Abbreviations: NC, negative control miRNA.

[0083] FIG. 3. 1 x 106 H226 cells were electroporated with 1.6 M hsa-let-7b or negative control miRNA (NC) and grown in standard growth media (day 0). On days 6, 10 and 17, cells were counted and repeatedly electroporated with 1.6 M
miRNA
(indicated by arrowheads). To accommodate exponential cell growth, a fraction of the total cell population was re-seeded after miRNA delivery on days 10 and 17. Cell counts were extrapolated and plotted onto a linear scale. The graph shows one representative experiment.

[0084] FIG. 4. Percent (%) proliferation of H460 lung cancer cells following administration of various combinations of microRNAs. A positive sign under each bar in the graph indicates that the microRNA was present in the administered combination. Synergistic activity of two microRNAs is indicated by the letter "S"
under the bar; additive activity of two microRNAs is indicated by the letter "A" under the bar. Standard deviations are shown in the graph. Abbreviations: Etopo, etoposide; NC, negative control miRNA.

[0085] FIG. 5. Average tumor volumes in mice harboring xenografts of A549 lung cancer cells treated with hsa-let-7b or with a negative control (NC) miRNA.
Standard deviations are shown in the graph. Data points with p values <0.05 are indicated by an asterisk.

DETAILED DESCRIPTION OF THE INVENTION

[0086] The present invention is directed to compositions and methods relating to the identification and characterization of genes and biological pathways related to these genes as represented by the expression of the identified genes, as well as use of miRNAs related to such, for therapeutic, prognostic, and diagnostic applications, particularly those methods and compositions related to assessing and/or identifying pathological conditions directly or indirectly related to let-7 expression or the aberrant expression thereof.

[0087] In certain aspects, the invention is directed to methods for the assessment, analysis, and/or therapy of a cell or subject where certain genes have a reduced expression (relative to normal) as a result of an increased or decreased expression of the any one or a combination of let-7 family members (7a-1, 7a-2, 7a-3, 7b, 7c, 7d, 7e, 7f-1, 7f-2, 7g, and/or 7i) and/or genes with an increased expression (relative to normal) as a result of an increased or decreased expression of one or a combination of let-7 family members (7a-1, 7a-2, 7a-3, 7b, 7c, 7d, 7e, 7f-1, 7f-2, 7g, and/or 7i). The expression profile and/or response to let-7 expression or lack of expression are indicative of an individual with a pathological condition, e.g., cancer.

[0088] Prognostic assays featuring any one or combination of the miRNAs listed or the markers listed (including nucleic acids representative thereof) could be used to assess a patient to determine what if any treatment regimen is justified. As with the diagnostic assays mentioned above, the absolute values that define low expression will depend on the platform used to measure the miRNA(s). The same methods described for the diagnostic assays could be used for a prognostic assays.

I. THERAPEUTIC METHODS

[0089] Embodiments of the invention concern nucleic acids that perform the activities of or inhibit endogenous miRNAs when introduced into cells. In certain aspects, nucleic acids are synthetic or non-synthetic miRNA. Sequence-specific miRNA inhibitors can be used to inhibit sequentially or in combination the activities of one or more endogenous miRNAs in cells, as well those genes and associated pathways modulated by the endogenous miRNA.

[0090] The present invention concerns, in some embodiments, short nucleic acid molecules that function as miRNAs or as inhibitors of miRNA in a cell. The term "short" refers to a length of a single polynucleotide that is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 100, or 150 nucleotides or fewer, including all integers or ranges derivable there between. The nucleic acid molecules are typically synthetic.
The term "synthetic" refers to a nucleic acid molecule that is isolated and not produced naturally in a cell. In certain aspects the sequence (the entire sequence) and/or chemical structure deviates from a naturally-occurring nucleic acid molecule, such as an endogenous precursor miRNA or miRNA molecule or complement thereof. While in some embodiments, nucleic acids of the invention do not have an entire sequence that is identical or complementary to a sequence of a naturally-occurring nucleic acid, such molecules may encompass all or part of a naturally-occurring sequence or a complement thereof. It is contemplated, however, that a synthetic nucleic acid administered to a cell may subsequently be modified or altered in the cell such that its structure or sequence is the same as non-synthetic or naturally occurring nucleic acid, such as a mature miRNA sequence. For example, a synthetic nucleic acid may have a sequence that differs from the sequence of a precursor miRNA, but that sequence may be altered once in a cell to be the same as an endogenous, processed miRNA or an inhibitor thereof. The term "isolated" means that the nucleic acid molecules of the invention are initially separated from different (in terms of sequence or structure) and unwanted nucleic acid molecules such that a population of isolated nucleic acids is at least about 90% homogenous, and may be at least about 95, 96, 97, 98, 99, or 100%
homogenous with respect to other polynucleotide molecules. In many embodiments of the invention, a nucleic acid is isolated by virtue of it having been synthesized in vitro separate from endogenous nucleic acids in a cell. It will be understood, however, that isolated nucleic acids may be subsequently mixed or pooled together.
In certain aspects, synthetic miRNA of the invention are RNA or RNA analogs.
miRNA inliibitors may be DNA or RNA, or analogs thereof miRNA and miRNA
inhibitors of the invention are collectively referred to as "synthetic nucleic acids."
[0091] In some embodiments, there is a miRNA or a synthetic miRNA having a length of between 17 and 130 residues. The present invention concerns miRNA or synthetic miRNA molecules that are, are at least, or are at most 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 140, 145, 150, 160, 170, 180, 190, 200 or more residues in length, including any integer or any range there between.

[0092] In certain embodiments, synthetic miRNA have (a) an "miRNA region"
whose sequence or binding region from 5' to 3' is identical or complementary to all or a segment of a mature miRNA sequence, and (b) a "complementary region" whose sequence from 5' to 3' is between 60% and 100% complementary to the miRNA
sequence in (a). In certain embodiments, these synthetic miRNA are also isolated, as defined above. The term "miRNA region" refers to a region on the synthetic miRNA
that is at least 75, 80, 85, 90, 95, or 100% identical, including all integers there between, to the entire sequence of a mature, naturally occurring miRNA
sequence or complement thereof. In certain embodiments, the miRNA region is or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% identical to the sequence of a naturally-occurring miRNA or complement thereof.

[0093] The term "complementary region" or "complement" refers to a region of a nucleic acid, mimetic, or synthetic miRNA that is or is at least 60%
complementary to the mature, naturally occurring miRNA sequence. The complementary region is or is at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any range derivable therein. With single polynucleotide sequences, there may be a hairpin loop structure as a result of chemical bonding between the miRNA region and the complementary region. In other embodiments, the complementary region is on a different nucleic acid molecule than the miRNA region, in which case the complementary region is on the complementary strand and the miRNA region is on the active strand.

[0094] In other embodiments of the invention, there are synthetic nucleic acids that are miRNA inhibitors. A miRNA inhibitor is between about 17 to 25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of a mature miRNA. In certain embodiments, a miRNA inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. Moreover, an miRNA inhibitor may have a sequence (from 5' to 3') that is or is at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any range derivable therein, to the 5' to 3' sequence of a mature miRNA, particularly a mature, naturally occurring miRNA. One of skill in the art could use a portion of the miRNA
sequence that is complementary to the sequence of a mature miRNA as the sequence for a miRNA inhibitor. Moreover, that portion of the nucleic acid or probe sequence can be altered so that it is still comprises the appropriate percentage of complementarity to the sequence of a mature miRNA.

[0095] In some embodiments, of the invention, a synthetic miRNA or inhibitor contains one or more design element(s). These design elements include, but are not limited to: (i) a replacement group for the phosphate or hydroxyl of the nucleotide at the 5' terminus of the complementary region; (ii) one or more sugar modifications in the first or last 1 to 6 residues of the complementary region; or, (iii) noncomplementarity between one or more nucleotides in the last 1 to 5 residues at the 3' end of the complementary region and the corresponding nucleotides of the miRNA
region. A variety of design modifications are known in the art, see below.

[0096] In certain embodiments, a synthetic miRNA has a nucleotide at its 5' end of the complementary region in which the phosphate and/or hydroxyl group has been replaced with another chemical group (referred to as the "replacement design"). In some cases, the phosphate group is replaced, while in others, the hydroxyl group has been replaced. In particular embodiments, the replacement group is biotin, an amine group, a lower alkylamine group, an acetyl group, 2'O-Me (2'oxygen-methyl), DMTO (4,4'-dimethoxytrityl with oxygen), fluoroscein, a thiol, or acridine, though other replacement groups are well known to those of skill in the art and can be used as well. This design element can also be used with a miRNA inhibitor.

[0097] Additional embodiments concern a synthetic miRNA having one or more sugar modifications in the first or last 1 to 6 residues of the complementary region (referred to as the "sugar replacement design"). In certain cases, there is one or more sugar modifications in the first 1, 2, 3, 4, 5, 6 or more residues of the complementary region, or any range derivable therein. In additional cases, there are one or more sugar modifications in the last 1, 2, 3, 4, 5, 6 or more residues of the complementary region, or any range derivable therein, have a sugar modification. It will be understood that the terms "first" and "last" are with respect to the order of residues from the 5' end to the 3' end of the region. In particular embodiments, the sugar modification is a 2'O-Me modification, a 2'F modification, a 2'H modification, a 2'amino modification, a 4'thioribose modification or a phosphorothioate modification on the carboxy group linked to the carbon at position 6'. In further embodiments, there are one or more sugar modifications in the first or last 2 to 4 residues of the complementary region or the first or last 4 to 6 residues of the complementary region.
This design element can also be used with a miRNA inhibitor. Thus, a miRNA
inhibitor can have this design element and/or a replacement group on the nucleotide at the 5' terminus, as discussed above.

[0098] In other embodiments of the invention, there is a synthetic miRNA or inhibitor in which one or more nucleotides in the last 1 to 5 residues at the 3' end of the complementary region are not complementary to the corresponding nucleotides of the miRNA region ("noncomplementarity") (referred to as the "noncomplementarity design"). The noncomplementarity may be in the last 1, 2, 3, 4, and/or 5 residues of the complementary miRNA. In certain embodiments, there is noncomplementarity with at least 2 nucleotides in the complementary region.

[0099] It is contemplated that synthetic miRNA of the invention have one or more of the replacement, sugar modification, or noncomplementarity designs. In certain cases, synthetic RNA molecules have two of them, while in others these molecules have all three designs in place.

[00100] The miRNA region and the complementary region may be on the same or separate polynucleotides. In cases in which they are contained on or in the same polynucleotide, the miRNA molecule will be considered a single polynucleotide.
In embodiments in which the different regions are on separate polynucleotides, the synthetic miRNA will be considered to be comprised of two polynucleotides.

[00101] When the RNA molecule is a single polynucleotide, there can be a linker region between the miRNA region and the complementary region. In some embodiments, the single polynucleotide is capable of forming a hairpin loop structure as a result of bonding between the miRNA region and the complementary region.
The linker constitutes the hairpin loop. It is contemplated that in some embodiments, the linker region is, is at least, or is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 residues in length, or any range derivable therein. In certain embodiments, the linker is between 3 and 30 residues (inclusive) in length.

[00102] In addition to having a miRNA or inhibitor region and a complementary region, there may be flanking sequences as well at either the 5' or 3' end of the region. In some embodiments, there is or is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides or more, or any range derivable therein, flanking one or both sides of these regions.

[00103] Methods of the invention include reducing or eliminating activity of one or more miRNAs in a cell comprising introducing into a cell a miRNA inhibitor (which may be described generally herein as an miRNA, so that a description of miRNA, where appropriate, also will refer to a miRNA inhibitor); or supplying or enhancing the activity of one or more miRNAs in a cell. The present invention also concerns inducing certain cellular characteristics by providing to a cell a particular nucleic acid, such as a specific synthetic miRNA molecule or a synthetic miRNA inhibitor molecule. However, in methods of the invention, the miRNA molecule or miRNA
inhibitor need not be synthetic. They may have a sequence that is identical to a naturally occurring miRNA or they may not have any design modifications. In certain embodiments, the miRNA molecule and/or the miRNA inhibitor are synthetic, as discussed above.

[00104] The particular nucleic acid molecule provided to the cell is understood to correspond to a particular miRNA in the cell, and thus, the miRNA in the cell is referred to as the "corresponding miRNA." In situations in which a named miRNA
molecule is introduced into a cell, the corresponding miRNA will be understood to be the induced or inhibited miRNA or induced or inhibited miRNA function. It is contemplated, however, that the miRNA molecule introduced into a cell is not a mature miRNA but is capable of becoming or functioning as a mature miRNA under the appropriate physiological conditions. In cases in which a particular corresponding miRNA is being inhibited by a miRNA inhibitor, the particular miRNA will be referred to as the "targeted miRNA." It is contemplated that multiple corresponding miRNAs may be involved. In particular embodiments, more than one miRNA
molecule is introduced into a cell. Moreover, in other embodiments, more than one miRNA inhibitor is introduced into a cell. Furthermore, a combination of miRNA
molecule(s) and miRNA inhibitor(s) may be introduced into a cell. The inventors contemplate that a combination of miRNA may act at one or more points in cellular pathways of cells with aberrant phenotypes and that such combination may have increased efficacy on the target cell while not adversely effecting normal cells. Thus, a combination of miRNA may have a minimal adverse effect on a subject or patient while supplying a sufficient therapeutic effect, such as amelioration of a condition, growth inhibition of a cell, death of a targeted cell, alteration of cell phenotype or physiology, slowing of cellular growth, sensitization to a second therapy, sensitization to a particular therapy, and the like.

[00105] Methods include identifying a cell or patient in need of inducing those cellular characteristics. Also, it will be understood that an amount of a synthetic nucleic acid that is provided to a cell or organism is an "effective amount,"
which refers to an amount needed (or a sufficient amount) to achieve a desired goal, such as inducing a particular cellular characteristic(s).

[00106] In certain embodiments of the methods include providing or introducing to a cell a nucleic acid molecule corresponding to a mature miRNA in the cell in an amount effective to achieve a desired physiological result.

[00107] Moreover, methods can involve providing synthetic or nonsynthetic miRNA molecules. It is contemplated that in these embodiments, that the methods may or may not be limited to providing only one or more synthetic miRNA
molecules or only one or more nonsynthetic miRNA molecules. Thus, in certain embodiments, methods may involve providing both synthetic and nonsynthetic miRNA molecules.
In this situation, a cell or cells are most likely provided a synthetic miRNA
molecule corresponding to a particular miRNA and a nonsynthetic miRNA molecule corresponding to a different miRNA. Furtherrnore, any method articulated using a list of miRNAs using Markush group language may be articulated without the Markush group language and a disjunctive article (i.e., or) instead, and vice versa.

[00108] In some embodiments, there is a method for reducing or inhibiting cell proliferation comprising introducing into or providing to the cell an effective amount of (i) a miRNA inhibitor molecule or (ii) a synthetic or nonsynthetic miRNA
molecule that corresponds to a miRNA sequence. In certain embodiments the methods involves introducing into the cell an effective amount of (i) an miRNA
inhibitor molecule having a 5' to 3' sequence that is at least 90%
complementary to the 5' to 3' sequence of one or more mature miRNA.

[00109] Certain embodiments of the invention include methods of treating a pathologic condition, in particular cancer, e.g., lung or liver cancer. In one aspect, the method comprises contacting a target cell with one or more nucleic acid, synthetic miRNA, or miRNA comprising at least one nucleic acid segment having all or a portion of a miRNA sequence. The segment may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides or nucleotide analog, including all integers there between. An aspect of the invention includes the modulation of gene expression, miRNA expression or function or mRNA expression or function within a target cell, such as a cancer cell.

[00110] Typically, an endogenous gene, miRNA or mRNA is modulated in the cell. In particular embodiments, the nucleic acid sequence comprises at least one segment that is at least 70, 75, 80, 85, 90, 95, or 100% identical in nucleic acid sequence to one or more miRNA or gene sequence. Modulation of the expression or processing of an endogenous gene, miRNA, or mRNA can be through modulation of the processing of a mRNA, such processing including transcription, transportation and/or translation with in a cell. Modulation may also be effected by the inhibition or enhancement of miRNA activity with a cell, tissue, or organ. Such processing may affect the expression of an encoded product or the stability of the mRNA. In still other embodiments, a nucleic acid sequence can comprise a modified nucleic acid sequence. In certain aspects, one or more miRNA sequence may include or comprise a modified nucleobase or nucleic acid sequence.

[00111] It will be understood in methods of the invention that a cell or other biological matter such as an organism (including patients) can be provided a miRNA
or miRNA molecule corresponding to a particular miRNA by administering to the cell or organism a nucleic acid molecule that functions as the corresponding miRNA
once inside the cell. The form of the molecule provided to the cell may not be the form that acts a miRNA once inside the cell. Thus, it is contemplated that in some embodiments, a synthetic miRNA or a nonsynthetic miRNA is provided a synthetic miRNA or a nonsynthetic miRNA, such as one that becomes processed into a mature and active miRNA once it has access to the cell's miRNA processing machinery.
In certain embodiments, it is specifically contemplated that the miRNA molecule provided to the biological matter is not a mature miRNA molecule but a nucleic acid molecule that can be processed into the mature miRNA once it is accessible to miRNA processing machinery. The term "nonsynthetic" in the context of miRNA
means that the miRNA is not "synthetic," as defined herein. Furthermore, it is contemplated that in embodiments of the invention that concern the use of synthetic miRNAs, the use of corresponding nonsynthetic miRNAs is also considered an aspect of the invention, and vice versa. It will be understand that the term "providing" an agent is used to include "administering" the agent to a patient.

[00112] In certain embodiments, methods also include targeting a miRNA to modulate in a cell or organism. The term "targeting a miRNA to modulate" means a nucleic acid of the invention will be employed so as to modulate the selected miRNA.
In some embodiments the modulation is achieved with a synthetic or non-synthetic miRNA that corresponds to the targeted miRNA, which effectively provides the targeted miRNA to the cell or organism (positive modulation). In other embodiments, the modulation is achieved with a miRNA inhibitor, which effectively inhibits the targeted miRNA in the cell or organism (negative modulation).

[00113] In some embodiments, the miRNA targeted to be modulated is a miRNA
that affects a disease, condition, or pathway. In certain embodiments, the miRNA is targeted because a treatment can be provided by negative modulation of the targeted miRNA. In other embodiments, the miRNA is targeted because a treatment can be provided by positive modulation of the targeted miRNA or its targets.

[00114] In certain methods of the invention, there is a further step of administering the selected miRNA modulator to a cell, tissue, organ, or organism (collectively "biological matter") in need of treatment related to modulation of the targeted miRNA
or in need of the physiological or biological results discussed herein (such as with respect to a particular cellular pathway or result like decrease in cell viability).
Consequently, in some methods of the invention there is a step of identifying a patient in need of treatment that can be provided by the miRNA modulator(s). It is contemplated that an effective amount of a miRNA modulator can be administered in some embodiments. In particular embodiments, there is a therapeutic benefit conferred on the biological matter, where a "therapeutic benefit" refers to an improvement in the one or more conditions or symptoms associated with a disease or condition or an improvement in the prognosis, duration, or status with respect to the disease. It is contemplated that a therapeutic benefit includes, but is not limited to, a decrease in pain, a decrease in morbidity, a decrease in a symptom. For example, with respect to cancer, it is contemplated that a therapeutic benefit can be inhibition of tumor growth, prevention of metastasis, reduction in number of metastases, inhibition of cancer cell proliferation, induction of cell death in cancer cells, inhibition of angiogenesis near cancer cells, induction of apoptosis of cancer cells, reduction in pain, reduction in risk of recurrence, induction of chemo- or radiosensitivity in cancer cells, prolongation of life, and/or delay of death directly or indirectly related to cancer.

[00115] Furthermore, it is contemplated that the miRNA compositions may be provided as part of a therapy to a patient, in conjunction with traditional therapies or preventative agents. Moreover, it is contemplated that any method discussed in the context of therapy may be applied as preventatively, particularly in a patient identified to be potentially in need of the therapy or at risk of the condition or disease for which a therapy is needed.

[00116] In addition, methods of the invention concern employing one or more nucleic acids corresponding to a miRNA and a therapeutic drug. The nucleic acid can enhance the effect or efficacy of the drug, reduce any side effects or toxicity, modify its bioavailability, and/or decrease the dosage or frequency needed. In certain embodiments, the therapeutic drug is a cancer therapeutic. Consequently, in some embodiments, there is a method of treating cancer in a patient comprising administering to the patient the cancer therapeutic and an effective amount of at least one miRNA molecule that improves the efficacy of the cancer therapeutic or protects non-cancer cells. Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments. Combination chemotherapies include but are not limited to, for example, 5-fluorouracil, alemtuzumab, amrubicin, bevacizumab, bleomycin, bortezomib, busulfan, camptothecin, capecitabine, cisplatin (CDDP), carboplatin, cetuximab, chlorambucil, cisplatin (CDDP), EGFR
inhibitors (gefitinib and cetuximab), procarbazine, mechlorethamine, cyclophosphamide, camptothecin, COX-2 inhibitors (e.g., celecoxib), cyclophosphamide, cytarabine, ) ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, dasatinib, daunorubicin, dexamethasone, docetaxel, doxorubicin (adriamycin), EGFR
inhibitors (gefitinib and cetuximab), erlotinib, estrogen receptor binding agents, bleomycin, plicomycin, mitomycin, etoposide (VP16), everolimus, tamoxifen, raloxifene, estrogen receptor binding agents, taxol, taxotere, gemcitabien, navelbine, famesyl-protein transferase inhibitors, gefitinib, gemcitabine, gemtuzumab, ibritumomab, ifosfamide, imatinib mesylate, larotaxel, lapatinib, lonafarnib, mechlorethamine, melphalan, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, mitomycin, navelbine, nitrosurea, nocodazole, oxaliplatin, paclitaxel, plicomycin, procarbazine, raloxifene, rituximab, sirolimus, sorafenib, sunitinib, tamoxifen, taxol, taxotere, temsirolimus, tipifarnib, tositumomab, transplatinum, trastuzumab, vinblastin, vincristin, or vinorelbine or any analog or derivative variant of the foregoing.

[00117] Generally, inhibitors of miRNAs can be given to decrease the activity of an endogenous miRNA. For example, inhibitors of miRNA molecules that increase cell proliferation can be provided to cells to increase proliferation or inhibitors of such molecules can be provided to cells to decrease cell proliferation. The present invention contemplates these embodiments in the context of the different physiological effects observed with the different miRNA molecules and miRNA
inhibitors disclosed herein. These include, but are not limited to, the following physiological effects: increase and decreasing cell proliferation, increasing or decreasing apoptosis, increasing transformation, increasing or decreasing cell viability, activating or inhibiting a kinase (e.g., Erk)ERK, activating/inducing or inhibiting hTert, inhibit stimulation of growth promoting pathway (e.g., Stat signaling), reduce or increase viable cell number, and increase or decrease number of cells at a particular phase of the cell cycle. Methods of the invention are generally contemplated to include providing or introducing one or more different nucleic acid molecules corresponding to one or more different miRNA molecules. It is contemplated that the following, at least the following, or at most the following number of different nucleic acid or miRNA molecules may be provided or introduced:
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range derivable therein. This also applies to the number of different miRNA molecules that can be provided or introduced into a cell.

II. PHARMACEUTICAL FORMULATIONS AND DELIVERY

[00118] Methods of the present invention include the delivery of an effective amount of a miRNA or an expression construct encoding the same. An "effective amount" of the pharmaceutical composition, generally, is defined as that amount sufficient to detectably and repeatedly to achieve the stated desired result, for example, to ameliorate, reduce, minimize or limit the extent of the disease or its symptoms. Other more rigorous definitions may apply, including elimination, eradication or cure of disease.

A. Administration [00119] In certain embodiments, it is desired to kill cells, inhibit cell growth, inhibit metastasis, decrease tumor or tissue size, and/or reverse or reduce the malignant or disease phenotype of cells. The routes of administration will vary, naturally, with the location and nature of the lesion or site to be targeted, and include, e.g., intradermal, subcutaneous, regional, parenteral, intravenous, intramuscular, intranasal, systemic, and oral administration and formulation. Direct injection, intratumoral injection, or injection into tumor vasculature is specifically contemplated for discrete, solid, accessible tumors, or other accessible target areas.
Local, regional, or systemic administration also may be appropriate. For tumors of >4 cm, the volume to be administered will be about 4-10 ml (preferably 10 ml), while for tumors of <4 cm, a volume of about 1-3 ml will be used (preferably 3 ml).

[00120] Multiple injections delivered as a single dose comprise about 0.1 to about 0.5 ml volumes. Compositions of the invention may be administered in multiple injections to a tumor or a targeted site. In certain aspects, injections may be spaced at approximately 1 cm intervals.

[00121] In the case of surgical intervention, the present invention may be used preoperatively, to render an inoperable tumor subject to resection.
Alternatively, the present invention may be used at the time of surgery, and/or thereafter, to treat residual or metastatic disease. For example, a resected tumor bed may be injected or perfused with a formulation comprising a miRNA or combinations thereof.
Administration may be continued post-resection, for example, by leaving a catheter implanted at the site of the surgery. Periodic post-surgical treatment also is envisioned. Continuous perfusion of an expression construct or a viral construct also is contemplated.

[00122] Continuous administration also may be applied where appropriate, for example, where a tumor or other undesired affected area is excised and the tumor bed or targeted site is treated to eliminate residual, microscopic disease.
Delivery via syringe or catherization is contemplated. Such continuous perfusion may take place for a period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longer following the initiation of treatment. Generally, the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs.

[00123] Treatment regimens may vary as well and often depend on tumor type, tumor location, immune condition, target site, disease progression, and health and age of the patient. Certain tumor types will require more aggressive treatment.
The clinician will be best suited to make such decisions based on the known efficacy and toxicity (if any) of the therapeutic formulations.

[00124] In certain embodiments, the tumor or affected area being treated may not, at least initially, be resectable. Treatments with compositions of the invention may increase the resectability of the tumor due to shrinkage at the margins or by elimination of certain particularly invasive portions. Following treatments, resection may be possible. Additional treatments subsequent to resection may serve to eliminate microscopic residual disease at the tumor or targeted site.

[00125] Treatments may include various "unit doses." A unit dose is defined as containing a predetermined quantity of a therapeutic composition(s). The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. With respect to a viral component of the present invention, a unit dose may conveniently be described in terms of g or mg of miRNA or miRNA mimetic. Alternatively, the amount specified may be the amount administered as the average daily, average weekly, or average monthly dose.

[00126] miRNA can be administered to the patient in a dose or doses of about or of at least about 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 g or mg, or more, or any range derivable therein.
Alternatively, the amount specified may be the amount administered as the average daily, average weekly, or average monthly dose, or it may be expressed in terms of mg/kg, where kg refers to the weight of the patient and the mg is specified above. In other embodiments, the amount specified is any number discussed above but expressed as mg/m2 (with respect to tumor size or patient surface area).

B. Injectable Compositions and Formulations [00127] In some embodiments, the method for the delivery of a miRNA or an expression construct encoding such or combinations thereof is via systemic administration. However, the pharmaceutical compositions disclosed herein may also be administered parenterally, subcutaneously, directly, intratracheally, intravenously, intradermally, intramuscularly, or even intraperitoneally as described in U.S.
Patents 5,543,158; 5,641,515 and 5,399,363 (each specifically incorporated herein by reference in its entirety).

[00128] Injection of nucleic acids may be delivered by syringe or any other method used for injection of a solution, as long as the nucleic acid and any associated components can pass through the particular gauge of needle required for injection. A
syringe system has also been described for use in gene therapy that permits multiple injections of predetermined quantities of a solution precisely at any depth (U.S. Patent 5,846,225).

[00129] Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[00130] In certain formulations, a water-based formulation is employed while in others, it may be lipid-based. In particular embodiments of the invention, a composition comprising a tumor suppressor protein or a nucleic acid encoding the same is in a water-based formulation. In other embodiments, the formulation is lipid based.

[00131] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral, intralesional, and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences"
15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

[00132] As used herein, a "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
Supplementary active ingredients can also be incorporated into the compositions.
[00133] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.

[00134] The nucleic acid(s) are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective.
The quantity to be administered depends on the subject to be treated, including, e.g., the aggressiveness of the disease or cancer, the size of any tumor(s) or lesions, the previous or other courses of treatment. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. Suitable regimes for initial administration and subsequent administration are also variable, but are typified by an initial administration followed by other administrations. Such administration may be systemic, as a single dose, continuous over a period of time spanning 10, 20, 30, 40, 50, 60 minutes, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and/or 1, 2, 3, 4, 5, 6, 7, days or more. Moreover, administration may be through a time release or sustained release mechanism, implemented by formulation and/or mode of administration.

C. Combination Treatments [00135] In certain embodiments, the compositions and methods of the present invention involve a miRNA, or expression construct encoding such. These miRNA
compositions can be used in combination with a second therapy to enhance the effect of the miRNA therapy, or increase the therapeutic effect of another therapy being employed. These compositions would be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and/or the inhibition of cellular hyperproliferation. This process may involve contacting the cells with the miRNA or second therapy at the same or different time. This may be achieved by contacting the cell with one or more compositions or pharmacological formulation that includes or more of the agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition provides (1) miRNA;
and/or (2) a second therapy. A second composition or method may be administered that includes a chemotherapy, radiotherapy, surgical therapy, immunotherapy, or gene therapy.

[00136] It is contemplated that one may provide a patient with the miRNA
therapy and the second therapy within about 12-24 h of each other and, more preferably, within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[00137] In certain embodiments, a course of treatment will last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 days or more. It is contemplated that one agent may be given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and/or 90, any combination thereof, and another agent is given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and/or 90, or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no treatment is administered.

This time period may last 1, 2, 3, 4, 5, 6, 7 days, and/or 1, 2, 3, 4, 5 weeks, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, depending on the condition of the patient, such as their prognosis, strength, health, etc.

[00138] Various combinations may be employed, for example miRNA therapy is "A" and a second therapy is "B":

[00139] A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/BB/B B/A/B/B
[00140] B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A
[00141] B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[00142] Administration of any compound or therapy of the present invention to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the vector or any protein or other agent.
Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described therapy.
[00143] In specific aspects, it is contemplated that a second therapy, such as chemotherapy, radiotherapy, immunotherapy, surgical therapy or other gene therapy, is employed in combination with the miRNA therapy, as described herein.

1. Chemotherapy [00144] A wide variety of chemotherapeutic agents may be used in accordance with the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. . Altennatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.

a. Alkylating agents [00145] Alkylating agents are drugs that directly interact with genomic DNA to prevent the cancer cell from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific. Alkylating agents can be implemented to treat chronic leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma, and particular cancers of the breast, lung, and ovary. They include: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan. Troglitazaone can be used to treat cancer in combination with any one or more of these alkylating agents.

b. Antimetabolites [00146] Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents, they specifically influence the cell cycle during S phase. They have been used to combat chronic leukemias in addition to tumors of breast, ovary and the gastrointestinal tract. Antimetabolites include 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate.

[00147] 5-Fluorouracil (5-FU) has the chemical name of 5-fluoro-2,4(1H,3H)-pyrimidinedione. Its mechanism of action is thought to be by blocking the methylation reaction of deoxyuridylic acid to thymidylic acid. Thus, 5-FU
interferes with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibits the formation of ribonucleic acid (RNA). Since DNA and RNA are essential for cell division and proliferation, it is thought that the effect of 5-FU is to create a thymidine deficiency leading to cell death. Thus, the effect of 5-FU is found in cells that rapidly divide, a characteristic of metastatic cancers.

c. Antitumor Antibiotics [00148] Antitumor antibiotics have both antimicrobial and cytotoxic activity.
These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes. These agents are not phase specific so they work in all phases of the cell cycle. Thus, they are widely used for a variety of cancers.
Examples of antitumor antibiotics include bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), and idarubicin, some of which are discussed in more detail below. Widely used in clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m2 at 21 day intervals for adriamycin, to 35-100 mg/m2 for etoposide intravenously or orally.

d. Mitotic Inhibitors [00149] Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis. They operate during a specific phase during the cell cycle. Mitotic inhibitors comprise docetaxel, etoposide (VP16), paclitaxel, taxol, taxotere, vinblastine, vincristine, and vinorelbine.
e. Nitrosureas [00150] Nitrosureas, like alkylating agents, inhibit DNA repair proteins. They are used to treat non-Hodgkin's lymphomas, multiple myeloma, and malignant melanoma, in addition to brain tumors. Examples include carmustine and lomustine.

2. Radiotherapy [00151] Radiotherapy, also called radiation therapy, is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated by damaging their genetic material, making it impossible for these cells to continue to grow. Although radiation damages both cancer cells and normal cells, the latter are able to repair them and function properly.
Radiotherapy may be used to treat localized solid tumors, such as cancers of the skin, tongue, larynx, brain, breast, or cervix. It can also be used to treat leukemia and lymphoma (cancers of the blood-forming cells and lymphatic system, respectively).
[00152] Radiation therapy used according to the present invention may include, but is not limited to, the use of y-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287) and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
Radiotherapy may comprise the use of radiolabeled antibodies to deliver doses of radiation directly to the cancer site (radioimmunotherapy). Once injected into the body, the antibodies actively seek out the cancer cells, which are destroyed by the cell-killing (cytotoxic) action of the radiation. This approach can minimize the risk of radiation damage to healthy cells.

[00153] Stereotactic radio-surgery (gamma knife) for brain and other tumors does not use a knife, but very precisely targeted beams of gamma radiotherapy from hundreds of different angles. Only one session of radiotherapy, taking about four to five hours, is needed. For this treatment a specially made metal frame is attached to the head. Then, several scans and x-rays are carried out to find the precise area where the treatment is needed. During the radiotherapy for brain tumors, the patient lies with their head in a large helmet, which has hundreds of holes in it to allow the radiotherapy beams through. Related approaches permit positioning for the treatment of tumors in other areas of the body.

3. Immunotherapy [00154] In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
Trastuzumab (HerceptinTM) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.
The combination of therapeutic modalities, i.e., direct cytotoxic activity and inhibition or reduction of ErbB2 would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers.

[00155] In one aspect of immunotherapy, the tumor or disease cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects.
Immune stimulating molecules also exist including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, and chemokines such as MIP-1, MCP-1, IL-8 and growth factors such as FLT3 ligand. Combining immune stimulating molecules, either as proteins or using gene delivery in combination with a tumor suppressor such as MDA-7 has been shown to enhance anti-tumor effects (Ju et al., 2000).
Moreover, antibodies against any of these compounds can be used to target the anti-cancer agents discussed herein.

[00156] Examples of immunotherapies currently under investigation or in use are immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998), cytokine therapy e.g., interferons a, (3 and y; IL-1, GM-CSF and TNF (Bukowski et al., 1998;
Davidson et al., 1998; Helistrand et al., 1998) gene therapy e.g., TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945) and monoclonal antibodies e.g., anti-ganglioside GM2, anti-HER-2, anti-p185; Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Patent 5,824,311).
Herceptin (trastuzumab) is a chimeric (mouse-human) monoclonal antibody that blocks the HER2-neu receptor. It possesses anti-tumor activity and has been approved for use in the treatment of malignant tumors (Dillman, 1999). A non-limiting list of several known anti-cancer immunotherapeutic agents and their targets includes (Generic Name (Target)) Cetuximab (EGFR), Panitumumab (EGFR), Trastuzumab (erbB2 receptor), Bevacizumab (VEGF), Alemtuzumab (CD52), Gemtuzumab ozogamicin (CD33), Rituximab (CD20), Tositumomab (CD20), Matuzumab (EGFR), Ibritumomab tiuxetan (CD20), Tositumomab (CD20), HuPAM4 (MUCI), MORAb-009 (Mesothelin), G250 (carbonic anhydrase IX), mAb 8H9 (8H9 antigen), M195 (CD33), Ipilimumab (CTLA4), HuLuc63 (CSl), Alemtuzumab (CD53), Epratuzumab (CD22), BC8 (CD45), HuJ591 (Prostate specific membrane antigen), hA20 (CD20), Lexatumumab (TRAIL receptor-2), Pertuzumab (HER-2 receptor), Mik-beta-1 (IL-2R), RAV12 (RAAG12), SGN-30 (CD30), AME-133v (CD20), HeFi-1 (CD30), BMS-663513 (CD137), Volociximab (anti-a5p1 integrin), GC1008 (TGF(3), HCD122 (CD40), Siplizumab (CD2), MORAb-003 (Folate receptor alpha), CNTO 328 (IL-6), MDX-060 (CD30), Ofatumumab (CD20), or SGN-33 (CD33). It is contemplated that one or more of these therapies may be employed with the miRNA therapies described herein.

[00157] A number of different approaches for passive immunotherapy of cancer exist. They may be broadly categorized into the following: injection of antibodies alone; injection of antibodies coupled to toxins or chemotherapeutic agents;
injection of antibodies coupled to radioactive isotopes; injection of anti-idiotype antibodies;
and finally, purging of tumor cells in bone marrow.

4. Gene Therapy [00158] In yet another embodiment, a combination treatment involves gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as one or more therapeutic miRNA. Delivery of a therapeutic polypeptide or encoding nucleic acid in conjunction with a miRNA may have a combined therapeutic effect on target tissues. A variety of proteins are encompassed within the invention, some of which are described below. Various genes that may be targeted for gene therapy of some form in combination with the present invention include, but are not limited to inducers of cellular proliferation, inhibitors of cellular proliferation, regulators of programmed cell death, cytokines and other therapeutic nucleic acids or nucleic acid that encode therapeutic proteins.

[00159] The tumor suppressor oncogenes function to inhibit excessive cellular proliferation. The inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation. The tumor suppressors (e.g., therapeutic polypeptides) p53, FHIT, p16 and C-CAM can be employed.

[00160] In addition to p53, another inhibitor of cellular proliferation is p16. The major transitions of the eukaryotic cell cycle are triggered by cyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4 (CDK4), regulates progression through the Gl. The activity of this enzyme may be to phosphorylate Rb at late GI. The activity of CDK4 is controlled by an activating subunit, D-type cyclin, and by an inhibitory subunit, the p16INK4 has been biochemically characterized as a protein that specifically binds to and inhibits CDK4, and thus may regulate Rb phosphorylation (Serrano et al., 1993; Serrano et al., 1995).
Since the p161NK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the activity of CDK4, resulting in hyperphosphorylation of the Rb protein.
p16 also is known to regulate the function of CDK6.

[00161] p16INK4 belongs to a newly described class of CDK-inhibitory proteins that also includes pl6B, p19, p21WAF1, and p27KIPl. The p16INK4 gene maps to 9p21, a chromosome region frequently deleted in many tumor types. Homozygous deletions and mutations of the p16INK4 gene are frequent in human tumor cell lines.
This evidence suggests that the p16INK4 gene is a tumor suppressor gene. This interpretation has been challenged, however, by the observation that the frequency of the p16INK4 gene alterations is much lower in primary uncultured tumors than in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994; Hussussian et al., 1994;
Kamb et al., 1994; Mori et al., 1994; Okamoto et al., 1994; Nobori et al., 1995;
Orlow et al., 1994; Arap et al., 1995). Restoration of wild-type p16INK4 function by transfection with a plasmid expression vector reduced colony formation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).

[00162] Other genes that may be employed according to the present invention include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zacl, p73, VHL, MMAC1 / PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p2l/p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fins, trk, ret, gsp, hst, abl, EIA, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors) and MCC.

5. Surgery [00163] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.

[00164] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[00165] Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
These treatments may be of varying dosages as well.

6. Other Agents [00166] It is contemplated that other agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines;
F42K and other cytokine analogs; or MIP-1, MIP-lbeta, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL (Apo-2 ligand) would potentiate the apoptotic inducing abilities of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.

[00167] Apo2 ligand (Apo2L, also called TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. TRAIL activates rapid apoptosis in many types of cancer cells, yet is not toxic to normal cells. TRAIL mRNA occurs in a wide variety of tissues. Most normal cells appear to be resistant to TRAIL's cytotoxic action, suggesting the existence of mechanisms that can protect against apoptosis induction by TRAIL. The first receptor described for TRAIL, called death receptor 4 (DR4), contains a cytoplasmic "death domain"; DR4 transmits the apoptosis signal carried by TRAIL. Additional receptors have been identified that bind to TRAIL.
One receptor, called DR5, contains a cytoplasmic death domain and signals apoptosis much like DR4. The DR4 and DR5 mRNAs are expressed in many normal tissues and tumor cell lines. Recently, decoy receptors such as DcRI and DcR2 have been identified that prevent TRAIL from inducing apoptosis through DR4 and DR5.
These decoy receptors thus represent a novel mechanism for regulating sensitivity to a pro-apoptotic cytokine directly at the cell's surface. The preferential expression of these inhibitory receptors in normal tissues suggests that TRAIL may be useful as an anticancer agent that induces apoptosis in cancer cells while sparing normal cells.
(Marsters et al., 1999).

[00168] There have been many advances in the therapy of cancer following the introduction of cytotoxic chemotherapeutic drugs. However, one of the consequences of chemotherapy is the development/acquisition of drug-resistant phenotypes and the development of multiple drug resistance. The development of drug resistance remains a major obstacle in the treatment of such tumors and therefore, there is an obvious need for alternative approaches such as gene therapy.

[00169] Another form of therapy for use in conjunction with chemotherapy, radiation therapy or biological therapy includes hyperthermia, which is a procedure in which a patient's tissue is exposed to high temperatures (up to 106 F).
External or internal heating devices may be involved in the application of local, regional, or whole-body hyperthermia. Local hyperthermia involves the application of heat to a small area, such as a tumor. Heat may be generated externally with high-frequency waves targeting a tumor from a device outside the body. Internal heat may involve a sterile probe , including thin, heated wires or hollow tubes filled with warm water, implanted microwave antennae, or radiofrequency electrodes.

[00170] A patient's organ or a limb is heated for regional therapy, which is accomplished using devices that produce high energy, such as magnets.
Alternatively, some of the patient's blood may be removed and heated before being perfused into an area that will be internally heated. Whole-body heating may also be implemented in cases where cancer has spread throughout the body. Warm-water blankets, hot wax, inductive coils, and thermal chambers may be used for this purpose.

[00171] Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.

[00172] This application incorporates U.S. Application Serial No. 11/349,727 filed on February 8, 2006 claiming priority to U.S. Provisional Application Serial No.
60/650,807 filed February 8, 2005 herein by references in its entirety.

III. MIRNA MOLECULES

[00173] MicroRNA molecules ("miRNAs") are generally 21 to 22 nucleotides in length, though lengths of 19 and up to 23 nucleotides have been reported. The miRNAs are each processed from a longer precursor RNA molecule ("precursor miRNA"). Precursor miRNAs are transcribed from non-protein-encoding genes. The precursor miRNAs have two regions of complementarity that enables them to form a stem-loop- or fold-back-like structure, which is cleaved in animals by a ribonuclease III-like nuclease enzyme called Dicer. The processed miRNA is typically a portion of the stem.

[00174] The processed miRNA (also referred to as "mature miRNA") becomes part of a large complex to down-regulate a particular target gene or its gene product.
Examples of animal miRNAs include those that imperfectly basepair with the target, which halts translation (Olsen et al., 1999; Seggerson et al., 2002). siRNA
molecules also are processed by Dicer, but from a long, double-stranded RNA molecule.
siRNAs are not naturally found in animal cells, but they can direct the sequence-specific cleavage of an mRNA target through a RNA-induced silencing complex (RISC) (Denli et al., 2003).

A. Array Preparation [00175] Certain embodiments of the present invention concerns the preparation and use of mRNA or nucleic acid arrays, miRNA or nucleic acid arrays, and/or miRNA
or nucleic acid probe arrays, which are macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary (over the length of the prove) or identical (over the length of the prove) to a plurality of nucleic acid, mRNA
or miRNA molecules, precursor miRNA molecules, or nucleic acids derived from the various genes and gene pathways modulated by let-7 miRNAs and that are positioned on a support or support material in a spatially separated organization.
Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted.
Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters.
Microarrays can be fabricated by spotting nucleic acid molecules, e.g., genes, oligonucleotides, etc., onto substrates or fabricating oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied in a high density matrix pattern of up to about 30 non-identical nucleic acid molecules per square centimeter or higher, e.g. up to about 100 or even 1000 per square centimeter.
Microarrays typically use coated glass as the solid support, in contrast to the nitrocellulose-based material of filter arrays. By having an ordered array of marker RNA and/or miRNA-complementing nucleic acid samples, the position of each sample can be tracked and linked to the original sample.

[00176] A variety of different array devices in which a plurality of distinct nucleic acid probes are stably associated with the surface of a solid support are known to those of skill in the art. Useful substrates for arrays include nylon, glass, metal, plastic, latex, and silicon. Such arrays may vary in a number of different ways, including average probe length, sequence or types of probes, nature of bond between the probe and the array surface, e.g. covalent or non-covalent, and the like.
The labeling and screening methods of the present invention and the arrays are not limited in its utility with respect to any parameter except that the probes detect miRNA, or genes or nucleic acid representative of genes; consequently, methods and compositions may be used with a variety of different types of nucleic acid arrays.
[00177] Representative methods and apparatus for preparing a microarray have been described, for example, in U.S. Patents 5,143,854; 5,202,231; 5,242,974;
5,288,644; 5,324,633; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807;
5,432,049; 5,436,327; 5,445,934; 5,468,613; 5,470,710; 5,472,672; 5,492,806;
5,525,464; 5,503,980; 5,510,270; 5,525,464; 5,527,681; 5,529,756; 5,532,128;
5,545,531; 5,547,839; 5,554,501; 5,556,752; 5,561,071; 5,571,639; 5,580,726;
5,580,732; 5,593,839; 5,599,695; 5,599,672; 5,610;287; 5,624,711; 5,631,134;
5,639,603; 5,654,413; 5,658,734; 5,661,028; 5,665,547; 5,667,972; 5,695,940;
5,700,637; 5,744,305; 5,800,992; 5,807,522; 5,830,645; 5,837,196; 5,871,928;
5,847,219; 5,876,932; 5,919,626; 6,004,755; 6,087,102; 6,368,799; 6,383,749;
6,617,112; 6,638,717; 6,720,138, as well as WO 93/17126; WO 95/11995; WO
95/21265; WO 95/21944; WO 95/35505; WO 96/31622; WO 97/10365; WO
97/27317; WO 99/35505; WO 09923256; WO 09936760; W00138580; WO
0168255; WO 03020898; WO 03040410; WO 03053586; WO 03087297; WO
03091426; W003100012; WO 04020085; WO 04027093; EP 373 203; EP 785 280;
EP 799 897 and UK 8 803 000; the disclosures of which are all herein incorporated by reference.

[00178] It is contemplated that the arrays can be high density arrays, such that they contain 2, 20, 25, 50, 80, 100 or more different probes. It is contemplated that they may contain 1000, 16,000, 65,000, 250,000 or 1,000,000 or more different probes.
The probes can be directed to mRNA and/or miRNA targets in one or more different organisms or cell types. The oligonucleotide probes range from 5 to 50, 5 to 45, 10 to 40, 9 to 34, or 15 to 40 nucleotides in length in some embodiments. In certain embodiments, the oligonucleotide probes are 5, 10, 15, 20 to 20, 25, 30, 35, nucleotides in length including all integers and ranges there between.

[00179] The location and sequence of each different probe sequence in the array are generally known. Moreover, the large number of different probes can occupy a relatively small area providing a high density array having a probe density of generally greater than about 60, 100, 600, 1000, 5,000, 10,000, 40,000, 100,000, or 400,000 different oligonucleotide probes per cm2. The surface area of the array can be about or less than about l, 1.6, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cmz.

[00180] Moreover, a person of ordinary skill in the art could readily analyze data generated using an array. Such protocols are disclosed above, and include information found in WO 9743450; WO 03023058; WO 03022421; WO 03029485;
WO 03067217; WO 03066906; WO 03076928; WO 03093810; WO 03100448A1, all of which are specifically incorporated by reference.

B. Sample Preparation [00181] It is contemplated that the RNA and/or miRNA of a wide variety of samples can be analyzed using the arrays, index of probes, or array technology of the invention. While endogenous miRNA is contemplated for use with compositions and methods of the invention, recombinant miRNA - including nucleic acids that are complementary or identical to endogenous miRNA or precursor miRNA - can also be handled and analyzed as described herein. Samples may be biological samples, in which case, they can be from biopsy, fine needle aspirates, exfoliates, blood, tissue, organs, semen, saliva, tears, other bodily fluid, hair follicles, skin, or any sample containing or constituting biological cells, particularly cancer or hyperproliferative cells. In certain embodiments, samples may be, but are not limited to, biopsy, or cells purified or enriched to some extent from a biopsy or other bodily fluids or tissues.
Alternatively, the sample may not be a biological sample, but be a chemical mixture, such as a cell-free reaction mixture (which may contain one or more biological enzymes).

C. Hybridization [00182] After an array or a set of probes is prepared and/or the nucleic acid in the sample or probe is labeled, the population of target nucleic acids is contacted with the array or probes under hybridization conditions, where such conditions can be adjusted, as desired, to provide for an optimum level of specificity in view of the particular assay being performed. Suitable hybridization conditions are well known to those of skill in the art and reviewed in Sambrook et al. (2001) and WO
95/21944. Of particular interest in many embodiments is the use of stringent conditions during hybridization. Stringent conditions are known to those of skill in the art.

[00183] It is specifically contemplated that a single array or set of probes may be contacted with multiple samples. The samples may be labeled with different labels to distinguish the samples. For example, a single array can be contacted with a tumor tissue sample labeled with Cy3, and normal tissue sample labeled with Cy5.
Differences between the samples for particular miRNAs corresponding to probes on the array can be readily ascertained and quantified.

[00184] The small surface area of the array permits uniform hybridization conditions, such as temperature regulation and salt content. Moreover, because of the small area occupied by the high density arrays, hybridization may be carried out in extremely small fluid volumes (e.g., about 250 l or less, including volumes of about or less than about 5, 10, 25, 50, 60, 70, 80, 90, 100 l, or any range derivable therein).
In small volumes, hybridization may proceed very rapidly.

D. Differential Expression Analyses [00185] Arrays of the invention can be used to detect differences between two samples. Specifically contemplated applications include identifying and/or quantifying differences between miRNA or gene expression from a sample that is normal and from a sample that is not normal, between a disease or condition and a cell not exhibiting such a disease or condition, or between two differently treated samples. Also, miRNA or gene expression may be compared between a sample believed to be susceptible to a particular disease or condition and one believed to be not susceptible or resistant to that disease or condition. A sample that is not normal is one exhibiting phenotypic or genotypic trait(s) of a disease or condition, or one believed to be not normal with respect to that disease or condition. It may be compared to a cell that is normal with respect to that disease or condition.
Phenotypic traits include symptoms of, or susceptibility to, a disease or condition of which a component is or may or may not be genetic, or caused by a hyperproliferative or neoplastic cell or cells.

[00186] An array comprises a solid support with nucleic acid probes attached to the support. Arrays typically comprise a plurality of different nucleic acid probes that are coupled to a surface of a substrate in different, known locations. These arrays, also described as "microarrays" or colloquially "chips" have been generally described in the art, for example, U.S. Patents 5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186 and Fodor et al., (1991), each of which is incorporated by reference in its entirety for all purposes. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Patent 5,384,261, incorporated herein by reference in its entirety for all purposes. Although a planar array surface is used in certain aspects, the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Patents 5,770,3 58, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, which are hereby incorporated in their entirety for all purposes.
Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all inclusive device, see for example, U.S. Patents 5,856,174 and 5,922,591 incorporated in their entirety by reference for all purposes. See also U.S.
patent application Ser. No. 09/545,207, filed April, 7, 2000 for additional information concerning arrays, their manufacture, and their characteristics, which is incorporated by reference in its entirety for all purposes.

[00187] Particularly, arrays can be used to evaluate samples with respect to pathological condition such as cancer and related conditions. It is specifically contemplated that the invention can be used to evaluate differences between stages or sub-classifications of disease, such as between benign, cancerous, and metastatic tissues or tumors.

[00188] Phenotypic traits to be assessed include characteristics such as longevity, morbidity, expected survival, susceptibility or receptivity to particular drugs or therapeutic treatments (drug efficacy), and risk of drug toxicity. Samples that differ in these phenotypic traits may also be evaluated using the compositions and methods described.

[00189] In certain embodiments, miRNA and/or expression profiles may be generated to evaluate and correlate those profiles with pharmacokinetics or therapies.
For example, these profiles may be created and evaluated for patient tumor and blood samples prior to the patient's being treated or during treatment to determine if there are miRNA or genes whose expression correlates with the outcome of the patient's treatment. Identification of differential miRNAs or genes can lead to a diagnostic assay for evaluation of tumor and/or blood samples to determine what drug regimen the patient should be provided. In addition, it can be used to identify or select patients suitable for a particular clinical trial. If an expression profile is determined to be correlated with drug efficacy or drug toxicity, that profile is relevant to whether that patient is an appropriate patient for receiving a drug, for receiving a combination of drugs, or for receiving a particular dosage of the drug.

[00190] In addition to the above prognostic assay, samples from patients with a variety of diseases can be evaluated to determine if different diseases can be identified based on miRNA and/or related gene expression levels. A diagnostic assay can be created based on the profiles that doctors can use to identify individuals with a disease or who are at risk to develop a disease. Alternatively, treatments can be designed based on miRNA profiling. Examples of such methods and compositions are described in the U.S. Provisional Patent Application entitled "Methods and Compositions Involving miRNA and miRNA Inhibitor Molecules" filed on May 23, 2005 in the names of David Brown, Lance Ford, Angie Cheng and Rich Jarvis, which is hereby incorporated by reference in its entirety.

E. Other Assays [00191) In addition to the use of arrays and microarrays, it is contemplated that a number of different assays could be employed to analyze miRNAs or related genes, their activities, and their effects. Such assays include, but are not limited to, nucleic acid amplification, polymerase chain reaction, quantitative PCR, RT-PCR, in situ hybridization, Northern hybridization, hybridization protection assay (HPA)(GenProbe), branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (US Genomics), Invader assay (ThirdWave Technologies), and/or Bridge Litigation Assay (Genaco).
IV. NUCLEIC ACIDS

[00192] The present invention concerns nucleic acids, modified or mimetic nucleic acids, miRNAs, mRNAs, genes, and representative fragments thereof that can be labeled, used in array analysis, or employed in diagnostic, therapeutic, or prognostic applications, particularly those related to pathological conditions such as cancer. The molecules may have been endogenously produced by a cell, or been synthesized or produced chemically or recombinantly. They may be isolated and/or purified.
Each of the miRNAs described herein and includes the corresponding SEQ ID NO and accession numbers for these miRNA sequences. The name of a miRNA is often abbreviated and referred to without a "hsa-" prefix and will be understood as such, depending on the context. Unless otherwise indicated, miRNAs referred to in the application are human sequences identified as miR-X or let-X, where X is a number and/or letter.

[00193] In certain aspects, a miRNA probe designated by a suffix "5P" or "3P"
can be used. "5P" indicates that the mature miRNA derives from the 5' end of the precursor and a corresponding "3P" indicates that it derives from the 3' end of the precursor, as described on the world wide web at sanger.ac.uk. Moreover, in some embodiments, a miRNA probe is used that does not correspond to a known human miRNA. It is contemplated that these non-human miRNA probes may be used in embodiments of the invention or that there may exist a human miRNA that is homologous to the non-human miRNA. In other embodiments, any mammalian cell, biological sample, or preparation thereof may be employed.

[00194] In some embodiments of the invention, methods and compositions involving miRNA may concern miRNA, markers (e.g., mRNAs), and/or other nucleic acids. Nucleic acids may be, be at least, or be at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides, or any range derivable therein, in length. Such lengths cover the lengths of processed miRNA, miRNA probes, precursor miRNA, miRNA containing vectors, mRNA, mRNA
probes, control nucleic acids, and other probes and primers.

[00195] In many embodiments, miRNA are 19-24 nucleotides in length, while miRNA probes are 19-35 nucleotides in length, depending on the length of the processed miRNA and any flanking regions added. miRNA precursors are generally between 62 and 110 nucleotides in humans.

[00196] Nucleic acids of the invention may have regions of identity or complementarity to another nucleic acid. It is contemplated that the region of complementarity or identity can be at least 5 contiguous residues, though it is specifically contemplated that the region is, is at least, or is at most 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 contiguous nucleotides. It is further understood that the length of complementarity within a precursor miRNA or other nucleic acid or between a miRNA probe and a miRNA or a miRNA gene are such lengths. Moreover, the complementarity may be expressed as a percentage, meaning that the complementarity between a probe and its target is 90% or greater over the length of the probe. In some embodiments, complementarity is or is at least 90%, 95% or 100%. In particular, such lengths may be applied to any nucleic acid comprising a nucleic acid sequence identified in any of SEQ ID NOs described herein, accession number, or any other sequence disclosed herein. Typically, the commonly used name of the miRNA is given (with its identifying source in the prefix, for example, "hsa"
for human sequences) and the processed miRNA sequence. Unless otherwise indicated, a miRNA without a prefix will be understood to refer to a human miRNA.
Moreover, a lowercase letter in a miRNA name may or may not be lowercase; for example, hsa-mir-130b can also be referred to as miR-130B. The term "miRNA
probe" refers to a nucleic acid probe that can identify a particular miRNA or structurally related miRNAs.

[00197] It is understood that some nucleic acids are derived from genomic sequences or a gene. In this respect, the term "gene" is used for simplicity to refer to the genomic sequence encoding the precursor nucleic acid or miRNA for a given miRNA or gene. However, embodiments of the invention may involve genomic sequences of a miRNA that are involved in its expression, such as a promoter or other regulatory sequences.

[00198] The term "recombinant" may be used and this generally refers to a molecule that has been manipulated in vitro or that is a replicated or expressed product of such a molecule.

[00199] The term "nucleic acid" is well known in the art. A "nucleic acid" as used herein will generally refer to a molecule (one or more strands) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide," each as a subgenus of the term "nucleic acid."

[00200] The term "miRNA" generally refers to a single-stranded molecule, but in specific embodiments, molecules implemented in the invention will also encompass a region or an additional strand that is partially (between 10 and 50%
complementary across length of strand), substantially (greater than 50% but less than 100%
complementary across length of strand) or fully complementary to another region of the same single-stranded molecule or to another nucleic acid. Thus, miRNA
nucleic acids may encompass a molecule that comprises one or more complementary or self-complementary strand(s) or "complement(s)" of a particular sequence. For example, precursor miRNA may have a self-complementary region, which is up to 100%
complementary. miRNA probes or nucleic acids of the invention can include, can be or can be at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100%
complementary to their target.

[00201] It is understood that a "synthetic nucleic acid" of the invention means that the nucleic acid does not have all or part of a chemical structure or sequence of a naturally occurring nucleic acid. Consequently, it will be understood that the term "synthetic miRNA" refers to a "synthetic nucleic acid" that functions in a cell or under physiological conditions as a naturally occurring miRNA.

[00202] While embodiments of the invention may involve synthetic miRNAs or synthetic nucleic acids, in some embodiments of the invention, the nucleic acid molecule(s) need not be "synthetic." In certain embodiments, a non-synthetic nucleic acid or miRNA employed in methods and compositions of the invention may have the entire sequence and structure of a naturally occurring mRNA or miRNA precursor or the mature mRNA or miRNA. For example, non-synthetic miRNAs used in methods and compositions of the invention may not have one or more modified nucleotides or nucleotide analogs. In these embodiments, the non-synthetic miRNA may or may not be recombinantly produced. In particular embodiments, the nucleic acid in methods and/or compositions of the invention is specifically a synthetic miRNA and not a non-synthetic miRNA (that is, not a miRNA that qualifies as "synthetic"); though in other embodiments, the invention specifically involves a non-synthetic miRNA and not a synthetic miRNA. Any embodiments discussed with respect to the use of synthetic miRNAs can be applied with respect to non-synthetic miRNAs, and vice versa.

[00203] It will be understood that the term "naturally occurring" refers to something found in an organism without any intervention by a person; it could refer to a naturally-occurring wildtype or mutant molecule. In some embodiments a synthetic miRNA molecule does not have the sequence of a naturally occurring miRNA
molecule. In other embodiments, a synthetic miRNA molecule may have the sequence of a naturally occurring miRNA molecule, but the chemical structure of the molecule, particularly in the part unrelated specifically to the precise sequence (non-sequence chemical structure) differs from chemical structure of the naturally occurring miRNA molecule with that sequence. In some cases, the synthetic miRNA
has both a sequence and non-sequence chemical structure that are not found in a naturally-occurring miRNA. Moreover, the sequence of the synthetic molecules will identify which miRNA is effectively being provided or inhibited; the endogenous miRNA will be referred to as the "corresponding miRNA." Corresponding miRNA
sequences that can be used in the context of the invention include, but are not limited to, all or a portion of those sequences in the SEQ IDs provided herein, as well as any other miRNA sequence, miRNA precursor sequence, or any sequence complementary thereof. In some embodiments, the sequence is or is derived from or contains all or part of a sequence identified herein to target a particular miRNA (or set of miRNAs) that can be used with that sequence. Any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or any number or range of sequences there between may be selected to the exclusion of all non-selected sequences.

[00204] As used herein, "hybridization", "hybridizes" or "capable of hybridizing"
is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature. The term "anneal" as used herein is synonymous with "hybridize." The term "hybridization", "hybridize(s)" or "capable of hybridizing" encompasses the terms "stringent condition(s)" or "high stringency" and the terrns "low stringency" or "low stringency condition(s)."

[00205] As used herein "stringent condition(s)" or "high stringency" are those conditions that allow hybridization between or within one or more nucleic acid strand(s) containing complementary sequence(s), but preclude hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segrnent thereof, and the like.

[00206] Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.5 M NaCl at temperatures of about 42 C to about 70 C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of formamide, tetramethylammonium chloride or other solvent(s) in a hybridization mixture.

[00207] It is also understood that these ranges, compositions and conditions for hybridization are mentioned by way of non-limiting examples only, and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison to one or more positive or negative controls.
Depending on the application envisioned it is preferred to employ varying conditions of hybridization to achieve varying degrees of selectivity of a nucleic acid towards a target sequence. In a non-limiting example, identification or isolation of a related target nucleic acid that does not hybridize to a nucleic acid under stringent conditions may be achieved by hybridization at low temperature and/or high ionic strength. Such conditions are termed "low stringency" or "low stringency conditions," and non-limiting examples of low stringency include hybridization performed at about 0.15 M
to about 0.9 M NaCI at a temperature range of about 20 C to about 50 C. Of course, it is within the skill of one in the art to further modify the low or high stringency conditions to suite a particular application.

A. Nucleobase, Nucleoside, Nucleotide, and Modified Nucleotides [00208] As used herein a "nucleobase" refers to a heterocyclic base, such as for example a naturally occurring nucleobase (i.e., an A, T, G, C or U) found in at least one naturally occurring nucleic acid (i.e., DNA and RNA), and naturally or non-naturally occurring derivative(s) and analogs of such a nucleobase. A
nucleobase generally can form one or more hydrogen bonds ("anneal" or "hybridize") with at least one naturally occurring nucleobase in a manner that may substitute for naturally occurring nucleobase pairing (e.g., the hydrogen bonding between A and T, G
and C, and A and U).

[00209] "Purine" and/or "pyrimidine" nucleobase(s) encompass naturally occurring purine and/or pyrimidine nucleobases and also derivative(s) and analog(s) thereof, including but not limited to, those a purine or pyrimidine substituted by one or more of an alkyl, caboxyalkyl, amino, hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol or alkylthiol moiety. Preferred alkyl (e.g., alkyl, carboxyalkyl, etc.) moieties comprise of from about 1, about 2, about 3, about 4, about 5, to about 6 carbon atoms. Other non-limiting examples of a purine or pyrimidine include a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil, a xanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, a bromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a 8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a 5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a 5-chlorouracil, a 5-propyluracil, a thiouracil, a 2-methyladenine, a methylthioadenine, a N,N-diemethyladenine, an azaadenines, a 8-bromoadenine, a hydroxyadenine, a 6-hydroxyaminopurine, a 6-thiopurine, a 4-(6-aminohexyl/cytosine), and the like. Other examples are well known to those of skill in the art.

[00210] As used herein, a "nucleoside" refers to an individual chemical unit comprising a nucleobase covalently attached to a nucleobase linker moiety. A
non-limiting example of a "nucleobase linker moiety" is a sugar comprising 5-carbon atoms (i.e., a "5-carbon sugar"), including but not limited to a deoxyribose, a ribose, an arabinose, or a derivative or an analog of a 5-carbon sugar. Non-limiting examples of a derivative or an analog of a 5-carbon sugar include a 2'-fluoro-2'-deoxyribose or a carbocyclic sugar where a carbon is substituted for an oxygen atom in the sugar ring.
Different types of covalent attachment(s) of a nucleobase to a nucleobase linker moiety are known in the art (Kornberg and Baker, 1992).

[00211] As used herein, a "nucleotide" refers to a nucleoside further comprising a "backbone moiety". A backbone moiety generally covalently attaches a nucleotide to another molecule comprising a nucleotide, or to another nucleotide to form a nucleic acid. The "backbone moiety" in naturally occurring nucleotides typically comprises a phosphorus moiety, which is covalently attached to a 5-carbon sugar. The attachment of the backbone moiety typically occurs at either the 3'- or 5'-position of the 5-carbon sugar. However, other types of attachments are known in the art, particularly when a nucleotide comprises derivatives or analogs of a naturally occurring 5-carbon sugar or phosphorus moiety.

[00212] A nucleic acid may comprise, or be composed entirely of, a derivative or analog of a nucleobase, a nucleobase linker moiety and/or backbone moiety that may be present in a naturally occurring nucleic acid. RNA with nucleic acid analogs may also be labeled according to methods of the invention. As used herein a "derivative"
refers to a chemically modified or altered form of a naturally occurring molecule, while the terms "mimic" or "analog" refer to a molecule that may or may not structurally resemble a naturally occurring molecule or moiety, but possesses similar functions. As used herein, a "moiety" generally refers to a smaller chemical or molecular component of a larger chemical or molecular structure. Nucleobase, nucleoside and nucleotide analogs or derivatives are well known in the art, and have been described (see for example, Scheit, 1980, incorporated herein by reference).

[00213] Additional non-limiting examples of nucleosides, nucleotides or nucleic acids include those in: U.S. Patents 5,681,947, 5,652,099 and 5,763,167, 5,614,617, 5,670,663, 5,872,232, 5,859,221, 5,446,137, 5,886,165, 5,714,606, 5,672,697, 5,466,786, 5,792,847, 5,223,618, 5,470,967, 5,378,825, 5,777,092, 5,623,070, 5,610,289, 5,602,240, 5,858,988, 5,214,136, 5,700,922, 5,708,154, 5,728,525, 5,637,683, 6,251,666, 5,480,980, and 5,728,525, each of which is incorporated herein by reference in its entirety.

[00214] Labeling methods and kits of the invention specifically contemplate the use of nucleotides that are both modified for attachment of a label and can be incorporated into a miRNA molecule. Such nucleotides include those that can be labeled with a dye, including a fluorescent dye, or with a molecule such as biotin.
Labeled nucleotides are readily available; they can be acquired commercially or they can be synthesized by reactions known to those of skill in the art.

[00215] Modified nucleotides for use in the invention are not naturally occurring nucleotides, but instead, refer to prepared nucleotides that have a reactive moiety on them. Specific reactive functionalities of interest include: amino, sulfhydryl, sulfoxyl, aminosulfhydryl, azido, epoxide, isothiocyanate, isocyanate, anhydride, monochlorotriazine, dichlorotriazine, mono-or dihalogen substituted pyridine, mono-or disubstituted diazine, maleimide, epoxide, aziridine, sulfonyl halide, acid halide, alkyl halide, aryl halide, alkylsulfonate, N-hydroxysuccinimide ester, imido ester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-propionamide, glyoxal, aldehyde, iodoacetyl, cyanomethyl ester, p-nitrophenyl ester, o-nitrophenyl ester, hydroxypyridine ester, carbonyl imidazole, and the other such chemical groups.
In some embodiments, the reactive functionality may be bonded directly to a nucleotide, or it may be bonded to the nucleotide through a linking group. The functional moiety and any linker cannot substantially impair the ability of the nucleotide to be added to the miRNA or to be labeled. Representative linking groups include carbon containing linking groups, typically ranging from about 2 to 18, usually from about 2 to 8 carbon atoms, where the carbon containing linking groups may or may not include one or more heteroatoms, e.g. S, 0, N etc., and may or may not include one or more sites of unsaturation. Of particular interest in many embodiments is alkyl linking groups, typically lower alkyl linking groups of I to 16, usually 1 to 4 carbon atoms, where the linking groups may include one or more sites of unsaturation. The functionalized nucleotides (or primers) used in the above methods of functionalized target generation may be fabricated using known protocols or purchased from commercial vendors, e.g., Sigma, Roche, Ambion, Biosearch Technologies and NEN. Functional groups may be prepared according to ways known to those of skill in the art, including the representative information found in U.S. Patents 4,404,289; 4,405,711;
4,337,063 and 5,268,486, and U.K.. Patent 1,529,202, which are all incorporated by reference.

[00216] Amine-modified nucleotides are used in several embodiments of the invention. The amine-modified nucleotide is a nucleotide that has a reactive amine group for attachment of the label. It is contemplated that any ribonucleotide (G, A, U, or C) or deoxyribonucleotide (G, A, T, or C) can be modified for labeling.
Examples include, but are not limited to, the following modified ribo- and deoxyribo-nucleotides: 5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and 8-[(6-amino)butyl]-amino-ATP; N6-(4-amino)butyl-ATP, N6-(6-amino)butyl-ATP, N4-[2,2-oxy bis-(ethylamine)]-CTP; N6-(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP; 5-propargylamino-CTP, 5-propargylamino-UTP; 5-(3-aminoallyl)-dUTP; 8-[(4-amino)butyl]-amino-dATP and 8-[(6-amino)butyl]-amino-dATP; N6-(4-amino)butyI-dATP, N6-(6-amino)butyl-dATP, N4-[2,2-oxy-bis-(ethylamine)]-dCTP;
N6-(6-Amino)hexyl-dATP; 8-[(6-Amino)hexyl]-amino-dATP; 5-propargylamino-dCTP, and 5-propargylamino-dUTP. Such nucleotides can be prepared according to methods known to those of skill in the art. Moreover, a person of ordinary skill in the art could prepare other nucleotide entities with the same amine-modification, such as a 5-(3-aminoallyl)-CTP, GTP, ATP, dCTP, dGTP, dTTP, or dUTP in place of a 5-(3-aminoallyl)-UTP.

B. Preparation of Nucleic Acids [00217] A nucleic acid may be made by any technique known to one of ordinary skill in the art, such as for example, chemical synthesis, enzymatic production, or biological production. It is specifically contemplated that miRNA probes of the invention are chemically synthesized.

[00218] In some embodiments of the invention, miRNAs are recovered or isolated from a biological sample. The miRNA may be recombinant or it may be natural or endogenous to the cell (produced from the cell's genome). It is contemplated that a biological sample may be treated in a way so as to enhance the recovery of small RNA molecules such as miRNA. U.S. Patent Application Serial No. 10/667,126 describes such methods and it is specifically incorporated by reference herein.
Generally, methods involve lysing cells with a solution having guanidinium and a detergent.

[00219] Alternatively, nucleic acid synthesis is performed according to standard methods. See, for example, Itakura and Riggs (1980) and U.S. Patents 4,704,362, 5,221,619, and 5,583,013, each of which is incorporated herein by reference.
Non-limiting examples of a synthetic nucleic acid (e.g., a synthetic oligonucleotide), include a nucleic acid made by in vitro chemically synthesis using phosphotriester, phosphite, or phosphoramidite chemistry and solid phase techniques such as described in EP 266,032, incorporated herein by reference, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al., 1986 and U.S.
Patent 5,705,629, each incorporated herein by reference. Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Patents 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.

[00220] A non-limiting example of an enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCRTM (see for example, U.S. Patents 4,683,202 and 4,682,195, each incorporated herein by reference), or the synthesis of an oligonucleotide described in U.S. Patent 5,645,897, incorporated herein by reference. See also Sambrook et al., 2001, incorporated herein by reference).

[00221] Oligonucleotide synthesis is well known to those of skill in the art.
Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Patents 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.

[00222] Recombinant methods for producing nucleic acids in a cell are well known to those of skill in the art. These include the use of vectors (viral and non-viral), plasmids, cosmids, and other vehicles for delivering a nucleic acid to a cell, which may be the target cell (e.g., a cancer cell) or simply a host cell (to produce large quantities of the desired RNA molecule). Alternatively, such vehicles can be used in the context of a cell free system so long as the reagents for generating the RNA
molecule are present. Such methods include those described in Sambrook, 2003, Sambrook, 2001 and Sambrook, 1989, which are hereby incorporated by reference.

C. Isolation of Nucleic Acids [00223] Nucleic acids may be isolated using techniques well known to those of skill in the art, though in particular embodiments, methods for isolating small nucleic acid molecules, and/or isolating RNA molecules can be employed. Chromatography is a process often used to separate or isolate nucleic acids from protein or from other nucleic acids. Such methods can involve electrophoresis with a gel matrix, filter columns, alcohol precipitation, and/or other chromatography. If miRNA from cells is to be used or evaluated, methods generally involve lysing the cells with a chaotropic (e.g., guanidinium isothiocyanate) and/or detergent (e.g., N-lauroyl sarcosine) prior to implementing processes for isolating particular populations of RNA.

[00224] In particular methods for separating miRNA from other nucleic acids, a gel matrix is prepared using polyacrylamide, though agarose can also be used. The gels may be graded by concentration or they may be uniform. Plates or tubing can be used to hold the gel matrix for electrophoresis. Usually one-dimensional electrophoresis is employed for the separation of nucleic acids. Plates are used to prepare a slab gel, while the tubing (glass or rubber, typically) can be used to prepare a tube gel. The phrase "tube electrophoresis" refers to the use of a tube or tubing, instead of plates, to form the gel. Materials for implementing tube electrophoresis can be readily prepared by a person of skill in the art or purchased, such as from C.B.S. Scientific Co., Inc. or Scie-Plas.

[00225] Methods may involve the use of organic solvents and/or alcohol to isolate nucleic acids, particularly miRNA used in methods and compositions of the invention.
Some embodiments are described in U.S. Patent Application Serial No.
10/667,126, which is hereby incorporated by reference. Generally, this disclosure provides methods for efficiently isolating small RNA molecules from cells comprising:
adding an alcohol solution to a cell lysate and applying the alcohol/lysate mixture to a solid support before eluting the RNA molecules from the solid support. In some embodiments, the arnount of alcohol added to a cell lysate achieves an alcohol concentration of about 55% to 60%. While different alcohols can be employed, ethanol works well. A solid support may be any structure, and it includes beads, filters, and columns, which may include a mineral or polymer support with electronegative groups. A glass fiber filter or column has worked particularly well for such isolation procedures.

[00226] In specific embodiments, miRNA isolation processes include: a) lysing cells in the sample with a lysing solution comprising guanidinium, wherein a lysate with a concentration of at least about I M guanidinium is produced; b) extracting miRNA molecules from the lysate with an extraction solution comprising phenol;
c) adding to the lysate an alcohol solution for forming a lysate/alcohol mixture, wherein the concentration of alcohol in the mixture is between about 35% to about 70%;
d) applying the lysate/alcohol mixture to a solid support; e) eluting the miRNA
molecules from the solid support with an ionic solution; and, f) capturing the miRNA
molecules. Typically the sample is dried and resuspended in a liquid and volume appropriate for subsequent manipulation.

V. LABELS AND LABELING TECHNIQUES

[00227] In some embodiments, the present invention concerns miRNA that are labeled. It is contemplated that miRNA may first be isolated and/or purified prior to labeling. This may achieve a reaction that more efficiently labels the miRNA, as opposed to other RNA in a sample in which the miRNA is not isolated or purified prior to labeling. In many embodiments of the invention, the label is non-radioactive.
Generally, nucleic acids may be labeled by adding labeled nucleotides (one-step process) or adding nucleotides and labeling the added nucleotides (two-step process).
A. Labeling Techniques [00228] In some embodiments, nucleic acids are labeled by catalytically adding to the nucleic acid an already labeled nucleotide or nucleotides. One or more labeled nucleotides can be added to miRNA molecules. See U.S. Patent 6,723,509, which is hereby incorporated by reference.

[00229] In other embodiments, an unlabeled nucleotide or nucleotides is catalytically added to a miRNA, and the unlabeled nucleotide is modified with a chemical moiety that enables it to be subsequently labeled. In embodiments of the invention, the chemical moiety is a reactive amine such that the nucleotide is an amine-modified nucleotide. Examples of amine-modified nucleotides are well known to those of skill in the art, many being commercially available such as from Ambion, Sigma, Jena Bioscience, and TriLink.

[00230] In contrast to labeling of cDNA during its synthesis, the issue for labeling miRNA is how to label the already existing molecule. The present invention concerns the use of an enzyme capable of using a di- or tri-phosphate ribonucleotide or deoxyribonucleotide as a substrate for its addition to a miRNA. Moreover, in specific embodiments, it involves using a modified di- or tri-phosphate ribonucleotide, which is added to the 3' end of a miRNA. Enzymes capable of adding such nucleotides include, but are not limited to, poly(A) polymerase, terminal transferase, and polynucleotide phosphorylase. In specific embodiments of the invention, a ligase is contemplated as not being the enzyme used to add the label, and instead, a non-ligase enzyme is employed. Terminal transferase catalyzes the addition of nucleotides to the 3' ternlinus of a nucleic acid. Polynucleotide phosphorylase can polymerize nucleotide diphosphates without the need for a primer.

B. Labels [00231] Labels on miRNA or miRNA probes may be colorimetric (includes visible and UV spectrum, including fluorescent), luminescent, enzymatic, or positron emitting (including radioactive). The label may be detected directly or indirectly.

Radioactive labels include 1251, 32P, 33p, and 35S. Examples of enzymatic labels include alkaline phosphatase, luciferase, horseradish peroxidase, and (3-galactosidase.
Labels can also be proteins with luminescent properties, e.g., green fluorescent protein and phycoerythrin.

[00232] The colorimetric and fluorescent labels contemplated for use as conjugates include, but are not limited to, Alexa Fluor dyes, BODIPY dyes, such as BODIPY
FL;
Cascade Blue; Cascade Yellow; coumarin and its derivatives, such as 7-amino-4-methylcoumarin, aminocoumarin and hydroxycoumarin; cyanine dyes, such as Cy3 and Cy5; eosins and erythrosins; fluorescein and its derivatives, such as fluorescein isothiocyanate; macrocyclic chelates of lanthanide ions, such as Quantum DyeTM;
Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red, tetramethylrhodamine and rhodamine 6G; Texas Red; , fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer; and, TOTAB.

[00233] Specific examples of dyes include, but are not limited to, those identified above and the following: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750; amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY
530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR, and, BODIPY-TR; Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, 2',4',5',7'-Tetrabromosulfonefluorescein, and TET.

[00234] Specific examples of fluorescently labeled ribonucleotides are available from Molecular Probes, and these include, Alexa Fluor 488-5-UTP, Fluorescein-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP, Tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, and BODIPY TR-14-UTP. Other fluorescent ribonucleotides are available from Amersham Biosciences, such as Cy3-UTP and Cy5-UTP.

[00235] Examples of fluorescently labeled deoxyribonucleotides include Dinitrophenyl (DNP)-11-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPY TMR-14-dUTP, Tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630/650-14-dUTP, BODIPY 650/665-14-dUTP; Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor 546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA-dCTP, Alexa Fluor 647-12-OBEA-dCTP.

[00236] It is contemplated that nucleic acids may be labeled with two different labels. Furthermore, fluorescence resonance energy transfer (FRET) may be employed in methods of the invention (e.g., Klostermeier et al., 2002;
Emptage, 2001;
Didenko, 2001, each incorporated by reference).

[00237] Alternatively, the label may not be detectable per se, but indirectly detectable or allowing for the isolation or separation of the targeted nucleic acid. For example, the label could be biotin, digoxigenin, polyvalent cations, chelator groups and the other ligands, include ligands for an antibody.

C. Visualization Techniques [00238] A number of techniques for visualizing or detecting labeled nucleic acids are readily available. Such techniques include, microscopy, arrays, Fluorometry, Light cyclers or other real time PCR machines, FACS analysis, scintillation counters, Phosphoimagers, Geiger counters, MRI, CAT, antibody-based detection methods (Westerns, immunofluorescence, immunohistochemistry), histochemical techniques, HPLC (Griffey et al., 1997), spectroscopy, capillary gel electrophoresis (Cummins et al., 1996), spectroscopy; mass spectroscopy; radiological techniques; and mass balance techniques.

[00239] When two or more differentially colored labels are employed, fluorescent resonance energy transfer (FRET) techniques may be employed to characterize association of one or more nucleic acid. Furthermore, a person of ordinary skill in the art is well aware of ways of visualizing, identifying, and characterizing labeled nucleic acids, and accordingly, such protocols may be used as part of the invention.

WO 2008/073922 PCTlUS2007/087037 Examples of tools that may be used also include fluorescent microscopy, a BioAnalyzer, a plate reader, Storm (Molecular Dynamics), Array Scanner, FACS
(fluorescent activated cell sorter), or any instrument that has the ability to excite and detect a fluorescent molecule.

VI. KITS

[00240] Any of the compositions described herein may be comprised in a kit. In a non-limiting example, reagents for isolating miRNA, labeling miRNA, and/or evaluating a miRNA population using an array, nucleic acid amplification, and/or hybridization can be included in a kit, as well reagents for preparation of samples from blood samples. The kit may further include reagents for creating or synthesizing miRNA probes. The kits will thus comprise, in suitable container means, an enzyme for labeling the miRNA by incorporating labeled nucleotide or unlabeled nucleotides that are subsequently labeled. In certain aspects, the kit can include amplification reagents. In other aspects, the kit may include various supports, such as glass, nylon, polymeric beads, and the like, and/or reagents for coupling any probes and/or target nucleic acids. It may also include one or more buffers, such as reaction buffer, labeling buffer, washing buffer, or a hybridization buffer, compounds for preparing the miRNA probes, and components for isolating miRNA. Other kits of the invention may include components for making a nucleic acid array comprising miRNA, and thus, may include, for example, a solid support.

[00241] Kits for implementing methods of the invention described herein are specifically contemplated. In some embodiments, there are kits for preparing miRNA
for multi-labeling and kits for preparing miRNA probes and/or miRNA arrays. In these embodiments, kit comprise, in suitable container means, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more of the following: (1) poly(A) polymerase; (2) unmodified nucleotides (G, A, T, C, and/or U); (3) a modified nucleotide (labeled or unlabeled);
(4) poly(A) polymerase buffer; and, (5) at least one microfilter; (6) label that can be attached to a nucleotide; (7) at least one miRNA probe; (8) reaction buffer;
(9) a miRNA array or components for making such an array; (10) acetic acid; (11) alcohol;
(12) solutions for preparing, isolating, enriching, and purifying miRNAs or miRNA
probes or arrays. Other reagents include those generally used for manipulating RNA, such as formamide, loading dye, ribonuclease inhibitors, and DNase.

[00242] In specific embodiments, kits of the invention include an array containing miRNA probes, as described in the application. An array may have probes corresponding to all known miRNAs of an organism or a particular tissue or organ in particular conditions, or to a subset of such probes. The subset of probes on arrays of the invention may be or include those identified as relevant to a particular diagnostic, therapeutic, or prognostic application. For example, the array may contain one or more probes that is indicative or suggestive of (1) a disease or condition (acute myeloid leukemia), (2) susceptibility or resistance to a particular drug or treatment;
(3) susceptibility to toxicity from a drug or substance; (4) the stage of development or severity of a disease or condition (prognosis); and (5) genetic predisposition to a disease or condition.

[00243] For any kit embodiment, including an array, there can be nucleic acid molecules that contain or can be used to amplify a sequence that is a variant of, identical to or complementary to all or part of any of SEQ IDs described herein. In certain embodiments, a kit or array of the invention can contain one or more probes for the miRNAs identified by the SEQ IDs described herein. Any nucleic acid discussed above may be implemented as part of a kit.

[00244] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
[00245] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.

[00246] However, the components of the kit may be provided as dried powder(s).
When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. In some embodiments, labeling dyes are provided as a dried power. It is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 g or at least or at most those amounts of dried dye are provided in kits of the invention. The dye may then be resuspended in any suitable solvent, such as DMSO.

[00247] Such kits may also include components that facilitate isolation of the labeled miRNA. It may also include components that preserve or maintain the miRNA or that protect against its degradation. Such components may be RNAse-free or protect against RNAses. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution.

[00248] A kit will also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.

[00249] Kits of the invention may also include one or more of the following:
Control RNA; nuclease-free water; RNase-free containers, such as 1.5 ml tubes;
RNase-free elution tubes; PEG or dextran; ethanol; acetic acid; sodium acetate;
ammonium acetate; guanidinium; detergent; nucleic acid size marker; RNase-free tube tips; and RNase or DNase inhibitors.

[00250] It is contemplated that such reagents are embodiments of kits of the invention. Such kits, however, are not limited to the particular items identified above and may include any reagent used for the manipulation or characterization of miRNA.
VII. EXAMPLES

[00251] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art. Unless otherwise designated, catalog numbers refer to products available by that number from Ambion, Inc. , The RNA Company.

METHODS FOR THE ANALYSIS OF GENE EXPRESSION FOLLOWING
MIRNA TRANSFECTION

[00252] Synthetic pre-miR miRNAs (Ambion) were reverse transfected into quadruplicate samples of A549, HepG2 or HL-60 cells. A549 and HepG2 cells were transfected using siPORT NeoFX (Ambion) according to the manufacturer's recommendations using the following parameters: 200,000 cells per well in a 6 well plate, 5.0 l of NeoFX, 30 nM final concentration of miRNAs in 2.5 ml. Cells were harvested at 72 hours post transfection. HL-60 cells were transfected using the Gene Pulser Xcell System (Bio-Rad Laboratories, Inc.; Hercules, CA, USA) according to the manufacturer's instructions and the following parameters: 500,000 cells per reaction in a volume of 150 1, in a 2-mm electroporation cuvette using a single square wave pulse of 25 msec.

[00253] Total RNA was extracted using RNAqueous-4PCR (Ambion) according to the manufacturer's reconunended protocol. mRNA array analyses were performed by Asuragen Services (Austin, TX), according to the company's standard operating procedures. Using the MessageAmpTM 11-96 aRNA Amplification Kit (Ambion, cat #1819), 2 g of total RNA were used for target preparation and labelling with biotin.
cRNA yields were quantified using an Agilent Bioanalyzer 2100 capillary electrophoresis protocol. Labeled target was hybridized to Affymetrix mRNA
arrays (Human HG-U133A 2.0 arrays) using the manufacturer's recommendations and the following parameters. Hybridizations were carried out at 45 C for 16 hours in an Affymetrix Model 640 hybridization oven. Arrays were washed and stained on an Affymetrix FS450 Fluidics station, running the wash script Midi_euk2v3 450.
The arrays were scanned on an Affymetrix GeneChip Scanner 3000. Summaries of the image signal data, group mean values, p-values with significance flags, log ratios and gene annotations for every gene on the array were generated using the Affymetrix Statistical Algorithm MAS 5.0 (GCOS v1.4). Data were normalized for the effect observed by the average of two negative control microRNA sequences and then were averaged together for presentation. The genes determined to be altered by treatment were detertnined by filtering all genes by fold-change relative to the two control transfections. Statistical significance was assessed by a t-test after the omnibus F-test was shown to be significant.

EXAMPLE 2:
GENE EXPRESSION ANALYSISIN A549. HEPG2. AND HL-60 CELLS

[00254] miRNAs are believed to primarily influence gene expression at the level of translation. However, it has recently been reported that in some instances, hsa-let-7 (Bagga et al., 2005) and other miRNAs (Lim et al., 2005) may reduce the mRNA
levels of direct targets, and such changes can be observed upon microarray gene expression analysis.

[00255] The experiments described here identify genes whose mRNA levels are affected by expression of hsa-let-7 in human lung cancer (A549), human liver cancer (HepG2) and human acute myeloid leukemia (HL-60) cell lines. A549, HepG2 or HL-60 cells were transfected with pre-miR hsa-let-7b (as a representative member of the hsa-let-7 miRNA family) as described in Example 1. The results of the microarray gene expression analyses are shown in Table 2, 3 and 4.

Table 2. Genes with altered mRNA expression levels in A549 cells, following transfection with pre-miR hsa-let-7b.
Gene S mbol RefSeq Fold Change y incor orated herein by reference in their entirety) 2'-PDE NM177966 -2.031 AADACLI NM020792 4.169 AASDHPPT NM_015423 -2.596 ACF NM_014576 /// NM138932 III NM 138933 -2.342 ACPL2 NM_152282 2.568 ACVRIB NM_004302 //I NM_020327 /// NM 020328 -2.020 ADA NM_000022 -2.614 ADAM12 NM_003474 /// NM021641 2.004 ADCY1 NM 021116 2.255 ADFP NM001122 2.038 AK5 NM_012093 /// NM_174858 2.096 AKAP2 /// PALM2- NM 001004065 /// NM 007203 NM 147150 3.107 ALCAM NM_001627 2.591 ALDH 1 A3 NM_000693 2.164 ALDH3A1 NM000691 2.814 ANGEL2 NM144567 -2.238 ANKRD22 NM_144590 2.134 ANKRD44 NM_153697 5.186 ANP32A NM_006305 -2.037 ANPEP NM_001150 3.502 ANXA8 NM001630 2.076 AOXl NM001159 2.132 AP 1 S 1 NM_001283 /// NM_057089 -2.326 AQP3 NM004925 2.111 ARF7 NM_025047 4.907 ARIDIA NM_006015 /// NM0 1 8450 /// NM_139135 -2.031 ARL6IP6 NM152522 -2.914 ARL7 NM005737 2.141 ASAM NM_024769 3.795 ASK NM_006716 -2.251 ASNS NM001673 /// NM_133436 /// NM183356 -2.206 ATF2 NM_001880 2.120 ATG 10 NM_031482 -2.333 ATPI 1C NM_001010986 /// NM_173694 2.676 ATP2B4 NM001001396 /// NM_001684 2.022 ATP6V1C1 NM_001007254 /// NM_001695 -2.311 ATP6V 1F NM004231 -2.102 ATP9A NM_006045 2.552 AURKB NM_004217 -2.989 AVEN NM_020371 -2.156 BAI2 NM_001703 2.098 BCATl NM005504 -2.520 BCCIP NM_016567 /// NM_078468 //I NM_078469 -2.304 BEX2 NM_032621 2.154 BEXLI XM_043653 2.181 BTF3L4 NM152265 -2.174 BTNL9 NM_152547 3.114 C10orf9 NM_145012 /// NM181698 -2.502 C 13orfl NM020456 -2.607 C 14orf111 NM015962 -2.044 C 14orf2 NM004894 -2.455 C14orf46 NM_001024674 -2.290 C16orf45 NM033201 2.342 C 17orf63 NM018182 -2.058 C 18orf17 NM153211 -2.153 C 18orP21 NM031446 -2.171 C 1 orfl 21 NM_016076 -2.042 Clorf139 NM001002292 /// NM_024911 2.025 Clorf24 NM022083 ///NM052966 2.225 C 1 orf25 NM_030934 -2.193 C1QDC1 NM 001002259 /// NM 023925 /// NM 032156 2.649 CIR NM_001733 2.135 C20orfl 00 NM_032883 3.548 C20orfl77 NM_022106 -2.455 C2orf32 NM015463 2.558 C3orfl7 NM_001025072 III NM_001025073 III NM_015412 -2.577 C6orfl2O NM_001029863 -2.347 C6orfl4l NM_153344 2.511 C6orf176 XM499048 -3.962 C6orf2ll NM024573 -2.278 C8orfl NM004337 2.660 C9orf125 NM032342 -3.962 C9orf3 NM032823 -2.289 CAMTAI NM_015215 2.232 CANTl NM138793 -2.128 CAPG NM_001747 2.353 CBX5 NM_012117 -3.718 CCL26 NM_006072 2.353 CCNA2 NM_001237 -2.474 CCNG2 NM_004354 2.005 CD164 NM_006016 -2.243 NM_000610 /// NM_001001389 /// NM_001001390 CD44 /// ~ 001001391 /// NM 001001392 2.071 NM 001025079 /// NM_001025080 /// NM 001777 CD47 /// ~ 198793 - 2.465 CD59 NM_000611 /// NM_203329 IlI NM_203330 III 2.185 CD9 NM_001769 2.197 CDA NM_001785 2.456 CDC25A NM_001789 /// NM_201567 -2.588 CDC34 NM_004359 -2.990 CDC42EP3 NM_006449 2.057 CDCPI NM_022842 !// NM178181 2.642 CDH19 NM_021153 2.019 CDK5R1 NM_003885 2.003 CDK8 NM_001260 -3.292 CEBPD NM 005195 -3.131 CFLAR NMu003879 2.273 CHD7 NM017780 -2.006 CHEKI NM 001274 -2.070 ChGn NM018371 2.090 CHIC i XR_000216 -2.169 CITED2 NM_006079 2.212 CLDN3 NM_001306 3.246 CLIC4 NM_013943 2.128 CNFN NM_032488 2.206 COL12A1 NM_004370 /// NM_080645 2.146 NM_005203 /// NM080798 /// NM_080799 /lI
COL13A1 NM 080800 /// NM 080801 NM 080802 3.227 COL4A5 NM000495 /// NM_033380 /// NM_033381 2.073 COL5A1 NM_000093 2.365 COL6A1 NM_001848 2.767 COL6A2 NM_001849 /// NM05 8 1 74 /// NM 058175 3.792 COR02B NM 006091 3.570 CPOX NM_000097 -2.163 CREB5 NM001011666 /// NM004904 /// NM_182898 2,031 CSDEI NM001007553 /// NM007158 -2.306 NM_006140 /// NM_172245 /// NM_172246 ///
CSF2RA NM 172247 /// NM 172248 /// NM 172249 2.365 CTPS NM001905 -2.212 CTPS2 NM019857 /// NM_175859 -2.346 CTSS NM_004079 2.570 CXCLI NM_001511 4.396 CXCL2 NM002089 4.868 CXCL3 NM_002090 4.152 CXCL5 NM_002994 3.654 CXorf45 NM_024810 3.034 CYP3A5 NM000777 2.050 CYR61 NM_001554 -2.818 DAF NM000574 2.590 DCAMKLI NM_004734 2.780 DDC NM000790 -4.408 DDX3Y NM004660 2.138 DGKA NM_001345 /// NM201444 /// NM201445 /// 2.187 DHX40 NM_024612 2.033 DIAPH2 NM_006729 /// NM_007309 -2.003 DICERl NM_030621 /// NM_177438 -4.505 DKFZp434J1015 XM_496849 /// XM_499257 2.216 DKFZp667M2411 NM_207323 2.475 DKK3 NM_0010180571// NM_013253 /// NM015881 3.449 DNER NM_139072 2.811 DOCK1 I NM_144658 2.066 DOCK2 NM_004946 2.488 DOCK9 NM015296 3.245 DPAGTI NM_001382 /// NM203316 -2.632 DPYSL4 NM_006426 2.671 DUSP16 NM_030640 -2.565 DUSP6 NM 00 1 946 1// NM_022652 2.124 E2F5 NM001951 -2.923 EDIL3 NM_005711 3.471 EGFL3 XM_031401 3.200 EGFL4 NM_001410 2.310 EHD 1 NM_006795 2.112 EHF NM_012153 2.530 EIF2C2 NM_012154 2.750 EIF4E3 NM_173359 2.081 ELF4 NM_001421 -2.175 ELOVL7 NM024930 3.863 EMP 1 NM_001423 2.211 EMP2 NM_001424 2.628 ENTPD7 NM_020354 2.157 EPHB2 NM004442 /// NM017449 2.018 EPLIN NM016357 2.303 ERO 1 L NM_014584 -3.927 EYA2 NM 005244 /// NM 172110 /// NM 172111 /// 2.022 NM_172112 /// NM172113 F2R NM_001992 3.514 F2RL2 NM_004101 -2.807 F5 NM_000130 -2.066 FAM54A NM_138419 -2.026 FAM61A NM_015578 2.251 FAM96A NM001014812 /// NM_032231 -2.015 FBNI NM 000138 2.407 FCGBP NM003890 4.974 FDXR NM_004110 /// NM024417 2.103 FGA NM_000508 /// NM021871 -3.480 FGB NM_005141 -5.014 FGFBP1 NM005130 2.232 FGFR4 NM_002011 /// NM022963 /// NM213647 -2.029 FGG NM_000509 /// NM021870 -2.461 FHL1 NM_001449 2.089 FHL2 NM_001450 /J/ NM_201555 /// NM_201556 2.036 FIGN NM_018086 -2.842 FLJ 10700 NM018182 -2.822 FLJ11259 NM_018370 3.624 FLJ20160 NM_017694 2.143 FLJ22313 NM022373 2.391 FLJ22833 NM_001031716 /// NM022837 2.060 FLJ30655 NM_144643 -2.028 FLJ36031 NM_175884 2.051 FLJ36748 NM_152406 2.337 FLJ39370 NM152400 3.186 FLJ43339 NM_207380 2.435 FLJ90709 NM173514 -2.574 FLRT3 NM013281 /// NM198391 -2.146 NM_001004417 /// NM_001004421 FMNL2 NM 001004422 2,219 /// N_M 052905 FOXO3A NM_001455 /// NM_201559 2.187 FOXQI NM_033260 3.010 FRMD6 NM_152330 2.959 FSTL1 NM007085 3.378 FVT1 NM_002035 2.179 FYN NM_002037 /// NM_153047 /// NM_153048 2.199 GALC NM000153 -2.359 GALE NM_000403 NM_001008216 -2.420 GALNACT-2 NM_018590 2.008 GALNTI2 NM_024642 2.588 GALNT2 NM_004481 -3.061 GARS NM002047 -2.134 GBP3 NM 018284 3.721 GCHI NM000161 /// NM_001024024 /// NM_001024070 2.242 GDA NM_004293 2.477 GEM1N7 NM001007269 /// NM_001007270 /// NM_024707 -2.505 GFPT2 NM_005110 2.175 GLB I NM 000404 -3.416 GLIPRI NM006851 2.448 GLUL NM 001033044 /// NM 001033056 /// NM 002065 2.125 GMNN NM_015895 -2.286 GNBI NM_002074 2.371 GNG2 NM_053064 2.067 GNG5 NM_005274 -2.613 GOLTIB NM_016072 -2.532 GTF2I NM_001518 /// NM_032999 /// NM_033000 /// -2,190 H1FX NM_006026 -2.033 H2BFS NM_017445 -2.382 HAS3 NM005329 /// NM_138612 2.710 HDHD 1 A NM_012080 -7.596 HERC4 NM_001017972 /// NM_015601 /// NM022079 3.356 HH114 NM032499 -2.084 HHIP NM_022475 2.009 HIPK3 NM_005734 2.241 HISTIH2BK NM_080593 -2.344 ~i NM 0334981// NM 033500 2'622 HLF NM_002126 2.501 HMGA2 NM001015886 /// NM003483 /// NM003484 -4.655 HMMR NM_0 12484 /// NM_012485 -3.297 HNRPC NM_004500 /// NM031314 -3.742 HOXA1 NM_005522 /// NM_153620 -2.535 HTRAl NM_002775 2.315 ICF45 NM_017872 2.015 IFI16 NM_005531 2.148 IFNEl NM176891 3.464 IGFBPI NM000596 /// NM001013029 2.598 IGFBP6 NM 002178 2.555 IL10RB NM000628 2.039 IL11 NM_000641 -2.922 IL17RD NM_017563 2.014 NM_001012631 /// NM_001012632 !!/
IL32 NM_001012633 2.085 IL8 NM_000584 7.197 ILF3 NM 004516 /// NM012218/// NM153464 2.143 IMP-1 NMu_006546 -2.676 IMP-2 NM_001007225 /// NM_006548 -2.534 INSIG1 NM_005542 /// NM_198336 //I NM198337 2.011 INSL4 NM_002195 -2.131 ITGA2 NM_002203 2.284 ITGA6 NM000210 2.013 ITGB4 NM_000213 /// NM_001005619 /// NM_001005731 3.702 ITGB8 NM_002214 2.362 ITPR2 NM002223 2.831 IVNSIABP NM_006469 /// NM_016389 2.978 JUB NM_032876 /// NM_198086 -2.858 JUN NM_002228 2.092 KCNJ16 NM_018658 /// NM_170741 1// NM_170742 -3.743 KCNKI NM 002245 2.064 KCNMAI NM_001014797 /// NM_002247 2.962 KCNN4 NM_002250 2.109 KDELCI NM_024089 2.341 KDELC2 NM 153705 2.794 KIAA0100 NM_014680 2.041 KIAA0179 NM_015056 -2.032 KIAA0507 -2.326 KIAA1287 NM_020748 -2.130 KIAA1462 XM_166132 2.220 KIAA1571 XM_371590 2.125 KIAA1641 NM020970 2.107 KIAA1702 --- -2.308 KIAA1815 NM_024896 2.031 KIAA1946 NM_177454 3.307 KIAA1971 XM_058720 2.057 KLF 11 NM_003597 3.448 KRT15 NM 002275 2.859 KRT19 NM002276 2.910 KYNU NM_001032998 /// NM_003937 -2.413 LICAM NM_000425 /// NM_024003 2.090 LAMB3 NM_000228 /// NM_001017402 2.156 LAMP2 NM_002294 /// NM_013995 2.548 LARP6 NM018357 /// NM_197958 2.025 LCAT NM_000229 2.127 LEPR NM001003679 J// NM_001003680 /// NM002303 -2.759 LEPROTLI NM_015344 -2.689 LGALS3 /// GALIG NM_002306 /// NM_194327 2.380 LGR4 NM_018490 -2.472 LGR6 NM_001017403 /// NM_001017404 /// NM_021636 2.447 LHFP NM_005780 2.520 LIN28B NM001004317 -6.529 LOC 116238 NM_138463 -2.322 LOC150759 XM_498456 /// XM_499585 2.713 LOC201651 XM114355 -2.280 LOC283464 XM_290597 -3.353 LOC284611 NM_001010883 2.128 LOC285513 NM_198281 -2.702 LOC285943 --- -2.652 LOC399959 XM_378316 2.719 LOC440737 XM_496446 2.720 LOC441027 XM 496707 2.910 LOC492304 NM001007139 2.604 LOC51315 NM_016618 2.208 LOC554202 --- 2.368 LOXL2 NM_002318 2.583 LPGAT1 NM014873 -2.141 LRP12 NM_013437 2.092 LSM6 NM_007080 -2.442 LTBP3 NM021070 2.405 LTBP4 NM003573 2.407 LYST NM_000081 /// NM_001005736 2.480 MAFF NM 012323 /// NM 152878 3.282 MAFK NM002360 -2.183 MAP3K9 NM_033141 -2.349 MARCH4 NM020814 2.430 MARS NM004990 -2.018 MCAM NM_006500 2.079 MDH2 NM_005918 -2.057 MED6 NM005466 -2.300 NM_001001651/// NM_001001653 ///
MED8 NM_001001654 -2.082 MGC 11102 NM_032325 -2.106 MGC 11308 NM_032889 -2.003 MGC13204 NM031465 -2.951 MGC 14289 NM_080660 -2.993 MGC18216 --- -2.441 MGC23909 NM_174909 -2.925 MGC2408 NM_032331 -2.471 MGC2560 NM_031452 -3.112 MICAL2 NM014632 2.124 MICB NM_005931 -3.386 MMP7 NM 002423 2.127 MNl NM002430 -2.037 M-RIP NM_015134 !// NM_201274 2.082 MRS2L NM020662 -2.336 MSRB3 NM_001031679 /// NM_198080 2.261 MT1E NM175617 2.263 MT1F NM_005949 2.583 MT1 G NM_005950 2.162 MT1H NM_005951 2.635 MT1M NM_176870 2.021 MT1X NM_005952 2.407 MT2A NM_005953 2.913 MTPN NM_145808 2.033 NM_001001924 /// NM_001001925 !//
MTUS 1 NM_001001927 -2.159 MUCSB XM_039877 3.213 MY01D NM015194 2.120 NANOS 1 NM_001009553 /// NM_199461 3.060 NAP 1 L1 NM004537 /// NM139207 -2.253 NAP 1 L3 NM_00453 8 2.028 NARG1 NM_057175 -2.372 NAV3 NM 014903 2.160 NDRGI NM~_006096 2.039 NDUFA5 NM005000 2.270 NEIL3 NM_018248 -2.344 NEK3 NM_002498 //J NM152720 -2.099 NEXN NM144573 2.374 NFIB NM_005596 2.161 NGEF NM 019850 2.259 NHSL1 XM~496826 2.602 NIDI NM_002508 13.062 NLN NM 020726 -2.344 NME4 NM_005009 -2.386 NME6 NM 005793 -2.601 NOV NM_002514 2.008 NPC2 NM_006432 2.065 NR2F6 NM_005234 -2.073 NRAS NM_002524 -3.277 NT5E NM002526 2.176 NUDT15 NM_018283 3.077 NUDT4 NM019094 /// NM199040 -2.548 NU1,98 NM0053871// NM016320 /// NM_139131 /// -3.260 OBSLl XM_051017 2.368 OLFM1 NM_0063341// NM_014279 /// NM058199 2.392 OSTbeta NM 178859 -3.403 P18SRP NM173829 -2.162 PABPC4 NM_003819 -2.222 PALM2-AKAP2 NM 007203 /// NM_147150 3.034 PANK3 NM024594 2.076 PAPOLA NM_032632 -2.205 PBEF1 NM_005746 ///NM_182790 2.004 PCTP NM_021213 -2.334 PDCD4 NM014456 /// NM145341 -2.068 PDE3A NM_000921 -2.340 NM_001011513 NM_001011514 PDLIMS NM 001011515 /// NM 001011516 /// NM 006457 2.028 PELI1 NM_020651 2.011 PGM2L1 NM 173582 -2.235 PGRMC 1 NM006667 -2.561 PHF19 NM_001009936 /// NM_015651 -2.126 PHLDAI NM_007350 2.276 PIGA NM002641 /// NM_020472 NM_020473 -2.240 PJA2 NM_014819 2.280 PLAGLI N4 002656 /// NM_006718 -2.228 PLAGL2 NM_002657 -2.732 PLAT NM_000930 NM_000931 /// NM_033011 2.110 PLAU NM_002658 3.175 PLCL2 NM015184 2.480 PLEKHH2 NM172069 2.299 PLSCR4 NM_020353 2.853 PODXL NM001018111 /// NM005397 3.975 POLR2D NM_004805 -2.153 PPARG NM 005037 /// NM_015869 /lI NM_138711 l// 2 074 NM~138712 PPFIAI N4 003626 /// NM177423 2.111 PPP1R15A NM_014330 2.075 PPP4C NM_002720 -2.099 PRICKLEI NM_153026 2.766 PRKAR2A NM_004157 -3.057 PRKCDBP NM_145040 3.685 PRP2 NM173490 2.459 PRRG4 NM_024081 2.910 PRSS 1PRSS2 NM_002769 /// NM_002770 //! NM_002771 2.040 PRSS3 NM_002771 4.893 PSME4 NM_014614 -3.059 PTRF NM012232 2.131 PTX1 NM016570 -2.305 PURB NM_033224 2.076 PYCARD NM_013258 /// NM_145182 /// NM_145183 2.136 NM_006775 /// NM 206853 /// NM 206854 Q~ NM 206855 3.366 RAB3B NM002867 3.180 RABEP2 NM_024816 2.453 RAGE NM_014226 2.470 RAP2B NM_002886 2.237 RASGEFIA NM_145313 3.000 RASSF2 NM_014737 //! NM_170773 /// NM170774 2.639 RBP4 NM 006744 2.172 NM_001008710 /// NM_001008711 ffl RBPMS NM001008712 -2.326 RBPMS2 NM_194272 2.002 RECK NM_021111 2.557 RGS2 NM002923 2.250 RHOB NM_004040 -2.028 RHOBTBI NM_001032380 //l NM_014836 //! NM_198225 -2.150 RIG --- 3.819 RIOK2 NM_018343 -2.405 RIS 1 NM_015444 5.424 RIT1 NM_006912 2.140 NM 007282 /// NM_183381 /// NM_183382 ///
RNF13 ~ 183383 /// NM 183384 -2.104 RNF 144 NM_014746 2.327 RNF 157 NM_052916 2.030 RNF182 NM_152737 3.611 RPS6KA5 NM_004755 /// NM_182398 2.203 RPUSD3 NM_173659 -3.416 RRM2B NM015713 2.230 RTCD 1 NM_003729 -2.683 RTN4IP 1 NM_032730 -2.594 RTN4RL2 NM_178570 -2.193 RUNX2 NM 001015051 /// NM_001024630 /1/ NM_004348 2.185 RY1 NM006857 2.393 S 100PBPR NM_001017406 /// NM022753 -2.194 SARIB NM_001033503 ///NM_016103 3.112 SAT NM002970 2.446 SCAMPI NM_004866 /// NM_052822 2.468 SCARA3 NM_016240 /// NM_182826 2.572 SCD5 NM_024906 2.401 SCEL NM_003843 /// NM_144777 -2.043 SCN1B NM_001037 /// NM_199037 2.728 SEC24A XM 094581 2.064 SEMA4B NM_~020210 /// NM_198925 2.978 NM 015129 /// NM_032569 /// NM_145799 ///
SEPT6 /// N PAC ~ 145800 /// NM 145802 2.002 SERP 1 NM 014445 -2.398 SERPINB9 NM_004155 -3.543 SERPINE2 NM_006216 3.237 SEZ6L2 NM_012410 /// NM_201575 2.092 SFRP1 NM_003012 2.192 SGK2 NM_016276 /// NM_170693 -2.167 SLC11A2 NM 000617 2.236 SLC16A2 NM_006517 2.756 SLC17A5 NM 012434 -2.453 SLC1A1 NM_004170 3.319 SLC22A4 NM_003059 2.499 SLC25A13 NM_014251 -2.391 SLC25A24 NM_013386 /// NM 213651 -2.411 SLC25A32 NM_030780 -2.231 SLC25A37 NM_016612 IlI NM 018579 2.030 SLC35D2 NM 007001 -2.779 SLC44A1 NM022109 IlI NM_080546 2.091 SLC4A11 NM032034 2.448 NM_021196 /// NM_033323 lll NM 133478 /1/
SLC4A5 NM 133479 - -2.439 SLC5A6 NM_021095 -2.965 SLC6A15 NM_018057 /// NM_182767 2.160 SLC6A6 NM_003043 2.012 SLC7A5 NM_003486 -2.555 SLC7A6 NM_003983 -2.172 SLC7A7 NM_003982 -2.071 SMAD2 NM_001003652 /// NM_005901 2.035 SMARCC 1 NM_003074 -2.107 SMURF2 NM_022739 3.642 SNAP23 NM_003825 /// NM 130798 -2.270 SNX5 N4 _014426 /// NM152227 -2.012 SOCS3 NM_003955 2.401 SOD2 NM_000636 /// NM_001024465 /// NM_001024466 2.039 SPCS3 NM_021928 -2.631 SPOCK NM_004598 2.958 SQRDL NM_021199 2.004 SRP46 NM032102 -2.244 SRPK2 NM 182691 /// NM_182692 2.012 SS18L1 NM~_015558 /// NM_198935 2.202 ST6GALNAC2 NM_006456 -2.414 STARD3NL NM032016 -2.311 STAT1 NM_007315 Il1 NM139266 -2.063 STC 1 NM_003155 2.166 STEAP3 NM_001008410 /// NM_018234 //l NM_182915 2.414 NM 003600 /Il NM_198433 IlI NM198434 lll STK6 NM 198435 /// NM 198436 /// NM 198437 -2'773 STRA6 NM_022369 2.165 STS-1 NM_032873 -2.023 SUSD2 NM_019601 2.326 SUV39H2 NM 024670 -2.173 SYNGR3 NMy_004209 2.531 SYT13 NM_020826 2.280 TAGLN NM_001001522 /// NM_003186 2.210 TBC1D2 NM 018421 2.085 WO 2008/073922 PCTlUS2007/087037 TBC1D7 NM_016495 2.136 TCEAL3 NM001006933 /// NM_032926 2.373 TDO2 NM_005651 2.248 TFAP2C NM003222 2.730 TFPI2 NM_006528 3.936 TFRC NM_003234 -2.539 TGFA NM_003236 2.602 TGFBRI NM004612 -2.453 THBS 1 NM_003246 -2.022 THEM4 NM_053055 /// NM_176853 -2.147 THUMPDI NM_017736 2.138 TIGAI NM_053000 -2.341 TK2 NM_004614 2.448 TKT NM001064 -2.520 TLN1 NM_006289 2.138 TM4SF20 NM_024795 -5.746 TMED5 NM_016040 -2.165 TMEM16A NM_018043 2.204 TMEM2 NM_013390 -2.525 TMEM50B NM_006134 2.193 TMEM87B NM_032824 2.282 TNFAIP3 NM_006290 2.275 TNFAIP6 NM007115 5.084 T'NFRSF 11 A NM_003839 2.148 TNFRSF25 NM_003790 /// NM_148965/// NM 148966 /// 2.002 NM 148967 /// NM 148968 /// NM~148969 TNNT 1 NM_003283 2.014 TNRC6A NM_014494 /// NM_020847 2.135 TOPIMT NM_052963 2.799 TRIM8 NM_030912 2.355 TSPAN5 NM005723 2.208 TSPYL5 NM_033512 -2.025 TTC7B NM_001010854 2.287 TTC9C NM_173810 -2.004 TWISTI NM_000474 2.353 UHMKI NM_175866 2.058 ULBP2 NM025217 3.094 VGCNLI NM_052867 2.307 VGL-3 NM_016206 -3.767 VPS33A NM_022916 -2.356 VPS54 NM_001005739 /// NM016516 -2.769 XDH NM_000379 3.375 XK NM_021083 -2.366 YES1 NM_005433 2.261 YWHAH NM_003405 3.251 ZC3H12C XM_370654 2.474 ZCCHC9 NM032280 -2.537 ZCSL2 NM_206831 -3.789 ZDHHC20 NM_153251 2.934 ZDHHC3 NM_016598 -2.172 ZFHXIB NM014795 3.267 ZNF294 NM 015565 -2.085 ZNF680 NM 178558 2.224 Table 3. Genes with altered mRNA expression levels in HepG2 cells, following transfection with re-miR hsa-let-7b.
Gene Symbol RefSeq Fold (incorporated herein by reference in their entirety) Change 2'-PDE NM_177966 -3.346 AADAC NM_001086 2.432 AADACLI NM_020792 2.175 AASDHPPT NM_015423 -2.081 ABCB10 NM_012089 -2.443 ABCC3 NM_003786 /// NM_020037 /// NM_020038 2.245 ABTI NM013375 -2.413 ACF NM_014576 /// NM_138932 /// NM138933 -2.141 ACVRIB NM_004302 /// NM 020327 /// NM_020328 -2.699 ACYP2 NM138448 2.082 ADCY7 NM_001114 2.676 ADH6 NM_000672 -2.172 AER61 NM173654 -2.171 AFAP NM_021638 NM198595 2.049 AGA NM000027 2.001 AGPS NM_003659 -2.047 AGTRI NM000685 /// NM_004835 /// NM_009585 NM_031850 /// 2.127 NM_032049 AGXT2L1 NM_031279 -2.445 AIG1 NM_016108 2.629 AK2 NM_001625 /// NM013411 -2.247 AKR1D1 NM_005989 -13.748 ALCAM NM_001627 2.286 ALDH3Al NM000691 16.662 ALDH9A1 NM 000696 2.105 AMPD3 NMu_000480 /// NM_001025389 /// NM_001025390 2.389 ANGPTLI NM_004673 2.022 ANKRD17 NM032217 /// NM_198889 -2.602 ANKRD32 NM032290 -2.668 ANP32A NM_006305 -2.046 ANP32E NM_030920 -2.028 ANXA3 NM005139 2.222 AOX1 NM_001159 2.232 APIN NM017855 -4.347 APOB NM_000384 -3.680 APOC3 NM_000040 XM_496537 -2.843 APP NM000484 NM_201413 NM_201414 2.774 AQP11 NM173039 2.381 AQP3 NM_004925 2.202 AQP8 NM_001169 2.442 ARG2 NM001172 2.069 ARID3A NM_005224 -2.839 ARID5B NM_032199 2.199 ARL5A NM012097 /// NM177985 -2.022 ARL6IP6 NM 152522 -3.416 ARL7 NM005737 3.082 ARL8 NM_178815 -2.383 ARMCX3 NM_016607 /// NM_177947/// NM177948 2.371 ARRDC3 NM_020801 2.928 ASCIZ NM_015251 2.427 ASH 1 L NM_018489 2.226 ASK NM_006716 -4.157 ASPH NM004318 /// NM 020164 /// NM_032466 NM_032467 /// 2.311 NM_032468 ATAD2 NM_014109 -3.130 ATP6VOA2 NM_012463 -2.109 ATP7B NM_000053 /// NM_001005918 -2.013 ATP8B3 NM_138813 2.446 ATP9A NM_006045 2.408 ATPAF 1 NM_022745 -2.127 ATRX NM000489 /// NM_138270 /// NM138271 2.115 AURKB NM_004217 -5.040 AXL NM_001699 /// NM_021913 2.796 AZGPI NM 001185 2.369 BAZ1A NM~_013448 ///NM_182648 -2.140 BAZ2B NM_013450 2.304 BCCIP NM_0 16567 /// NM_078468 /// NM_078469 -3.087 BIRC3 NM 001165 /// NM_182962 -2.491 BLVRA NMV000712 2.084 BLVRB NM_000713 2.394 BM039 NM_018455 -2.987 BMPR2 NM_001204 2.066 BNIP3L NM004331 2.241 BRCA1 NM007294 /// NM007295 /// NM_007296 /// NM 007297 /// -3.100 NM__007298 ~

BRCA2 NM000059 -3.286 BRIP1 NM 032043 -2.013 BRRN1 NM015341 -2.266 BST2 NM_004335 2.029 BTF3L4 NM_152265 -2.149 BTG 1 NM001731 2.292 BUB 1 NM_004336 -2.280 BUBIB NM_001211 -2.314 BXDC2 NM_018321 -2.367 BZRP NM_000714 /// NM_007311 2.936 ClOorflO NM_007021 3.682 C10orfil NM032024 2.105 ClOorf3 NM018131 -2.537 C10orf38 NM 001010924 2.289 ClOorf6 NM018121 -2.088 ClOorf9 NM145012 /// NM181698 -2.422 C13orf23 NM025138 /1/ NM170719 -2.698 C 14orf2 NM_004894 -2.044 C 14orf46 NM_001024674 -3.545 C14orf78 XM290629 3.185 C14orf94 NM017815 -2.021 C15orf23 NM033286 -2.128 C16orf45 NM 033201 2.182 C16orf52 NM_173501 2.334 C17orf27 NM020914 2.512 C18orf19 NM152352 -2.151 C18orf21 NM_031446 -2.129 C18orf24 NM145060 -2.872 C19orf33 NM033520 3.232 Clorf112 NM_018186 -2.649 Clorfl3l NM_152379 -2.087 Clorf135 NM_024037 -2.115 C 1 orf25 NM030934 -2.046 C l orf33 NM_016183 -2.093 C 1 orf55 NM152608 -2.042 C 1 orP85 NM_144580 2.077 C 1 S NM_001734 /// NM_201442 2.131 C2 NM_000063 2.093 C20orf112 NM080616 -2.117 C20or119 NM018474 2.131 C21orf45 NM_018944 -2.111 C2orfl7 NM_024293 2.063 C2orf3 NM_003203 -2.170 C3orf23 NM_001029839 /// NM_001029840 /// NM_173826 2.103 C4orf13 NM 001029998 NM001030316 /// NM_032128 -2.038 C4orf9 NM003703 -2.260 C5 NM001735 3.563 C6orf139 NM_018132 -2.640 C6orf2ll NM_024573 -2.048 C7orf23 NM_024315 -3.610 C8orfl NM 004337 2.629 C9orfl5O NM203403 2.195 C9orfl52 NM001012993 2.906 C9orf4O NM017998 -2.071 C9orf4l NM152420 -3.137 C9orf52 NM_152574 -2.479 C9orf76 NM_024945 -2.028 C9orf95 NM_017881 2.838 CACNA2D4 NM_001005737 /Il NM_001005766 NM_172364 2.297 CAMTAI NM_015215 2.015 CAPN2 NM_001748 2.097 CAV2 NM_001233 /// NM_198212 2.115 CCDC5 NM_138443 -2.022 CCNA2 NM_001237 -4.693 CCNB 1 NM_031966 -2.221 CCNE2 NM_057735 /// NM_057749 -3.087 CCNF NM_001761 -2.070 CCNG2 NM_004354 3.578 CCNJ NM_019084 -3.368 CCPG1 NM004748 /// NM020739 2.128 CD 109 NM133493 2.253 CD36 NM 000072 /// NM_001001547 /// NM001001548 2.002 CD58 NM_001779 2.081 CD59 NM_000611 /1/ NM_203329 /// NM 203330 /// NM_203331 2.188 CD7 NM 006137 2.042 CD9 NM_001769 4.674 CD99L2 NM031462 /// NM 134445 //! NM_134446 2.191 CDA NM_001785 3.653 CDC2 NM_001786 /// NM_033379 -2.199 CDC20 NM_001255 -2.172 CDC23 NM_004661 -2.419 CDC25A NM_001789 /// NM 201567 -8.007 CDC34 NM_004359 -2.829 CDC45L NM_003504 -2.495 CDC6 NM001254 -4.395 CDCAI NM_031423 /// NM_145697 -2.322 CDCA2 NM_152562 -2.917 CDCA3 NM 031299 -2.220 CDCA5 NM080668 -2.247 CDCA7 NM_031942 /// NM_145810 -3.452 CDCA8 NM_018101 -2.359 CDK2 NM_001798 /// NM052827 -2.069 CDK8 NM_001260 -2.537 CDKALl NM017774 -2.134 CDKN2B NM_004936 /// NM_078487 2.569 CDTI NM_030928 -2.913 CGO18 NM_052818 2.905 CGI-116 NM_016053 2.130 CHD6 NM_032221 2.017 CHD7 NM_017780 -2.384 CHEKl NM_001274 -3.280 ChGn NM018371 5.004 CHPF NM024536 2.615 CHST9 NM_031422 -2.248 CKSIB NM_001826 -2.437 CLIC3 NM_004669 4.155 CLTB NM_00 1834 NM 007097 2.099 COIL NM_004645 -2.160 COL4A5 NM_000495 /// NM_033380 /// NM_033381 3.563 COL4A6 NM_001847 /// NM_033641 2.124 COL6Al NM_001848 2.159 COL7A1 NM_000094 2.391 COTL1 NM021149 2.018 CPB2 NM_001872 /// NM_016413 -2.402 CPEB2 NM_182485 /// NM_182646 -2.193 CPOX NM_000097 -3.630 CPT1A NM_001031847 /// NM_001876 2.306 CREB3L2 NM_194071 -2.106 CREB5 NM_001011666 /// NM_004904 !// NM_182898 /// NM 182899 2.205 CRIP 1 NM_001311 2.474 CSPG6 NM_005445 -2.099 CTDSPL2 NM_016396 -2.289 CTPS NM001905 -2.733 CTSB NM_001908/// NM 147780 /// NM 147781 /// NM 147782 2.067 CTSC NM_001814 /// NM_148170 -2.180 CTSD NM_001909 2.244 CTTN NM_005231 /// NM_138565 2.692 CXorfl2 NM_003492 2.048 CXorfl5 NM_018360 -2.089 CXorf45 NM_024810 2.755 CXXl NM_003928 2.227 CXXC6 NM_030625 -2.424 CYGB NM_134268 3.437 CYLN2 NM003388 III NM_032421 2.479 CYP3A5 NM_000777 2.238 CYP3A7 NM_000765 2.963 CYP4F 11 NM_021187 2.318 CYP4F3 NM_000896 2.420 DAF NM_000574 2.418 DBNl NM_004395 /1/ NM_080881 2.361 DCCI NM_024094 -3.401 DCDC2 NM_016356 -2.019 DDC NM_000790 -4.057 DDX18 NM_006773 -2.225 DDX19A NM_018332 -2.032 DDX58 NM_014314 2.159 DENNDIA NM_020946///NM_024820 -2.026 DEPDCI NM_017779 -3.205 DEPDCIB NM_018369 -2.577 DFNA5 NM_004403 2.045 DGAT1 NM_012079 -2.129 DHFR NM_000791 -2.868 DICERI NM_030621 /// NM_177438 -6.058 DIO1 NM_000792 /// NM_213593 -4.696 DISC1 NM001012957 /// NM_0010129581// NM_0010129592.337 DKC I NM_001363 -2.271 DKFZp434B1231 NM_178275 2.069 DKFZp434J1015 XM_496849 /// XM_499257 2.004 DKFZp434NO35 NM_032262 2.077 DKK3 NM_001018057 /// NM_013253 /// NM_015881 2.244 DLCI NM_006094 IlI NM_024767 /// NM_182643 -2.088 DLEU2NM_006021 -2.452 BCMSUNL
DLG7 NM_014750 -2.223 DMD NM_000109 IlI NM_004006 /// NM_004007 /// NM_004009 III -2.648 NM_004010 DNA2L XM_166103 -2.547 DNAJB9 NM_012328 -2.347 DNAJC12 NM_021800///NM_201262 2.478 DNASE2 NM 001375 2.224 DOC1 NMV_014890 /// NM_182909 4.474 DOK6 NM_152721 2.547 DONSON NM_017613 /// NM_145794 /// NM 145795 -3.186 DOTIL NM_032482 -2.692 DPAGTI NM_001382 /// NM203316 -2.224 DPH5 NM_015958 -2,050 DST NM_001723 /// NM 015548 /// NM 020388 /// NM 183380 2.099 DTL NM_016448 -4.310 DTNA NM_001390 /// NM 001391 /// NM 001392 /// NM 032975 2.365 NM_032978 - - -DUSP7 NM_001947 -2.552 DUSP9 NM_001395 -5.552 DZIPI NM_014934 /// NM_198968 -2.582 E2F5 NM_001951 -4.074 E2F6 NM_001952 /// NM_198256 /// NM 198257 /lI NM_198258 /1/ -2.349 NM_198325 E2F8 NM_024680 -3.332 EAF2 NM_018456 -2.674 EGFL5 XM 376905 2.405 EGRI NM001964 -2.716 EIF2C2 NM_012154 -2.272 EIF2C4 NM_017629 4.100 EIF4E NM_001968 -2.069 Ellsl NM152793 2.372 ELOVL7 NM_024930 3.606 EMP2 NM001424 2.553 ENOSFI NM_017512 2.638 EPB41L5 NM_020909 -2.170 EPPK1 NM_031308 2.097 ERBB3 NM_001005915 /// NM001982 2.078 ERCCI NM_001983 /J/NM_202001 2.049 ERO 1 L NM_014584 -2.297 ESCO2 NM_001017420 -2.220 EXOSC8 NM_181503 -2.327 EZH2 NM_004456 /1/ NM152998 -2.605 F2R NM_001992 4.586 FABP 1 NM_001443 -4.106 FABP5 NM_001444 -2.278 FAM19A5 NM_015381 -2.176 FAM29A NM_017645 -2.176 FAM3B NM_058186 /// NM_206964 -2.097 FAM54A NM_138419 -3.764 FAM55C NM145037 2.106 FAM57A NM_024792 -2.319 FAM61A NM_015578 2.138 FAM72A NM_207418 -2.954 FANCD2 NM_0010181151// NM 033084 -2.722 FANCM NM_020937 -2.201 FBL NM_001436 -2.413 FBLIM1 NM_001024215 /// NM_001024216 NM_017556 2.093 FBXO25 NM 012173 /// NM_183420 /// NM_183421 -2.279 FBXO5 NM_012177 -2.306 FEN1 NM_004111 -2.334 FIBCD 1 NM_032843 2.122 FIGN NM_018086 -3.415 FIGNLI NM_022116 -2.272 FIPILI NM_030917 -2.116 FKSG14 NM 022145 -3.151 FLADI NM 025207 //1 NM_201398 -2.062 FLJ10038 --- 2.139 FLJ10292 NM_018048 -2.071 FLJ10534 NM_018128 -2.397 FLJ10700 NM_018182 -2.646 FLJ10719 NM_018193 -2.610 FLJ11000 NM 018295 2.120 FLJ11155 NM_018342 -2.201 FLJ11259 NM 018370 2.103 FLJ11273 NM018374 2.079 FLJ13391 NM_032181 2.258 FLJ13912 NM_022770 -2.405 FLJ20160 NM 017694 2.580 FLJ20364 NM017785 -2.375 FLJ20516 NM_017858 -3.084 FLJ20641 NM_017915 -2.229 FLJ20674 NM_019086 2.301 FLJ20719 XM_373827 /// XM_498427 2.043 FLJ21986 NM_024913 -6.726 FLJ22313 NM_022373 2.053 FLJ22624 NM_024808 -2.332 FLJ22833 NM_001031716 /// NM_022837 2.527 FLJ25371 NM_152543 -2.078 FLJ25416 NM_145018 -2.525 FLJ31306 XM_495990 2.300 FLJ31401 --- 2.150 FLJ32745 NM_144978 -2.927 FLJ34306 NM_199340 4.762 FLJ38725 NM_153218 2.003 FLJ39370 NM_152400 5.565 FLJ43339 NM_207380 2.195 FLJ90586 NM_153345 2.266 FMO5 NM_001461 2.184 FOSL2 NM_005253 2.797 FOXK2 NM_004514 /// NM_1 8 1430 /// NM_181431 -2.171 FOXO3A NM_001455 /// NM_201559 2.109 FTH1 NM_002032 2.011 FVT1 NM_002035 2.914 FZD3 NM_017412 -2.012 FZD6 NM_003506 2.277 G 1 P2 NM_005101 2.505 GIP3 NM_002038 /// NM_022872 /// NM_022873 2.180 G3BP NM_005754 /// NM_198395 -2.145 GABARAPLI NM031412 2.162 GAJ NM_032117 -4.247 GALE NM_000403 /// NM_001008216 -2.459 GALNACT-2 NM_018590 2.063 GALNS NM_000512 2.430 GART NM000819 /// NM_175085 -2.600 GBP2 NM_004120 2.543 GBP3 NM 018284 2.251 GDA NM_004293 2.723 GEMIN5 NM_015465 -2.127 GEMIN7 NM001007269 /// NM_001007270 /// NM_024707 -2.614 GIPC2 NM017655 -2.887 GK NM_000167 lll NM_203391 2.175 GLB 1 NM000404 -4.245 GLCCII NM_138426 2.065 GLCE NM_015554 2.101 GLIPRI NM_006851 2.047 GLS NM_014905 2.045 GMNN NM_015895 -3.074 GMPR2 NM_001002000 lll NM_001002001 lll NM_001002002 III -2.041 GNAI I NM_002069 5.503 GNB 1 NM_002074 2.579 GNB5 NM 006578 /// NM_016194 2.356 GNG5 NMv_005274 -2.407 GNS NM_002076 2.378 GPC I NM_002081 2.196 GPD 1 NM_005276 -2.324 GPR157 NM_024980 -2.905 GPR56 NM_005682 /lI NM_201524 lll NM201525 3.004 GRCC10 NM138425 2.526 GRN NM_001012479 /// NM_002087 2.237 GRPELI NM_025196 -2.752 GRPEL2 NM_152407 -2.219 GTPBP4 NM_012341 -2.005 GYG2 NM 003918 -2.029 H2AFY NM`004893 /// NM_138609 /// NM_138610 2.024 HBP 1 NM_012257 2.281 HCAP-G NM 022346 -2.785 HDHDIA NM012080 -5.292 HEAB NM_006831 -2.065 HELLS NM_018063 -2.791 HERC4 NM_001017972 lll NM_015601 Il1 NM_022079 2.566 HIC2 NM_015094 -4.228 HIPK3 NM_005734 3.158 HISTIHIC NM005319 2.202 HISTIH2AC NM_003512 2.999 HISTIH2BC NM_003526 2.256 HISTIH3H NM_003536 2.327 HIST2H2AA NM_003516 2.070 HIST2H2BE NM_003528 2.620 HIVEP2 NM_006734 2.040 HK1 NM_000188 IlI NM 033496 /// NM_033497 /// NM033498 III 2.452 NM 033500 ~
HMGA2 NM_001015886 /// NM003483 /// NM_003484 -8.387 HMGN4 NM_006353 2.049 HMMR NM_012484 /// NM012485 -5.557 HNRPC NM_004500 /// NM_031314 -3.426 HOMER3 NM_004838 2.278 HPCALI NM_002149 /// NM_134421 2.080 HPR NM_020995 -2.163 HRMTIL3 NM_005788 -2.125 HS2ST 1 NM_012262 2.233 HSA9761 NM_014473 -2.034 HSD17B2 NM_002153 2.103 HSPA14 NM_016299 -2.228 HSPB 1 NM_001540 2.727 HSPBB NM_014365 2.042 HSPC111 NM_016391 -2.381 HSPC159 NM_014181 2.698 HSUP I XM_497769 -2.085 ICAM2 NM_000873 3.025 IDS NM_000202 /// NM_006123 2.347 IF127 NM_005532 3.436 IFITM I NM003641 2.014 IFITM2 NM_006435 2.160 IGF2BP1 NM_006546 -2.943 IGFBPI NM 000596 /1/ NM_001013029 2.432 IGFBP4 NM001552 3.118 IGFBP7 NM_001553 2.208 IGSFl NM_001555 /// N1v1_205833 -2.245 IHPK2 NM001005909 /// NM_001005910 /// NM001005911 /// 2.163 NM_001005912 IL10RB NM_000628 2.826 IL1RN NM_000577 /!/ NM_173841 /// NM173842 /// N1vI_173843 2.004 IMP-1 NM_006546 -3.538 IMP-2 NM_001007225 /// NM 006548 -2.550 IMP4 NM 033416 -2.024 IP04 NM024658 -2.000 IP07 NM_006391 -2.053 IQCB1 NM 001023570 //! NM_001023571 -2.032 IRAK2 NM001570 -2.132 ISGF3G N1vI006084 2.804 ITGA2 NM002203 2.172 ITGA3 NM 002204 /// NM_005501 2.160 ITGB3BP NM_014288 -2.119 ITGB5 NM_002213 2.026 ITIH3 NM_002217 2.929 JDP2 NM_130469 2.459 KBTBD8 NM_032505 -3.346 KIAA0101 NM_001029989NM 014736 -2.203 KIAA0179 NM_015056 -2.486 KIAA0746 NM_015187 4.687 KIAA0802 NM_015210 2.240 KIAA0934 NM_014974 2.638 KIAAI199 NM_018689 2.008 KIAA1212 NM_018084 -2.021 KIAA1223 NM_020337 2.120 KIAA1287 NM_020748 -2.252 KIAA1458 XM_044434 -2.018 KIAA1462 XM_166132 -2.386 KIAA1609 NM 020947 -2.129 KIAA1618 NM020954 2.870 KIAA1702 --- -2.728 KIAA1815 NM_024896 2.258 KIF15 NM_020242 -2.249 KIF18A NM 031217 -2.257 KIF23 NM004856 NM_138555 -2.157 KIF3C NM002254 2.017 KIFC2 NM_145754 2.417 KLF11 NM 003597 3.040 KLHL14 NM~020805 -2.955 KLHL24 NMi_017644 2.327 KLHL9 NM_018847 2.043 KNS2 NM_005552 /// NM 182923 2.020 KNTC 1 NM_014708 -2.090 KRT 15 NM_002275 2.214 KRT20 NM_019010 13.981 KRT23 NM_015515 /// NM 173213 5.377 KRTAPI-5 NM_031957 2.295 KRTAP3-1 NM_031958 8.731 L3MBTL NM_015478 /// NM 032107 2.320 LAIR2 NM_002288 (/! NM021270 3.794 LAMB2 NM_002292 2.080 LARP6 NM_018357 NM_197958 2.924 LBR NM_002296 NM_194442 -2.387 LEAP-2 NM_052971 -2.118 LEPR NM_001003679 /// NM_001003680 /// NM002303 2.234 LEPROTL1 NM_015344 -2.321 LGALS 1 NM002305 2.299 LGALS2 NM006498 -4.968 LGALS3 /// GALIG NM_002306 /// NM 194327 2.547 LGALS7 NM_002307 3.311 LIN28B NM001004317 -12.185 LKAP NM014647 2.657 LMBRI NM_022458 2.066 LMNB I NM_005573 -2.717 LOC123876 NM_001010845 -2.100 LOC123876 NM 001010845 /// NM 182617 -2.039 LOC131076 NM001017928 -2.534 LOC144501 NM_182507 2.511 LOC145786 --- -6.142 LOC146909 XM_085634 -2.071 LOC153222 NM153607 2.772 LOC158563 --- -2.207 LOC159090 NM_145284 2.305 LOC162993 XM_091914 2.428 LOC201175 NM_174919 2.612 LOC201725 NM001008393 -2.950 LOC201895 NM_174921 2.177 LOC253842 -- -4.200 LOC283377 NM207344 -2.105 LOC283464 XM 290597 -2.824 LOC283666 --- 2.566 LOC283852 --- 2.149 LOC284356 --- 2.469 LOC285628 --- 2.027 LOC340061 NM_198282 2.116 LOC340109 XM_379322 2.256 LOC387921 NM_001012754 /// NM_001017370 -2.589 LOC389432 NM_001030060 3.174 LOC391020 XM_497663 2.015 LOC440461 XM_498680 2.303 LOC440702 XM_496425 2.036 LOC440737 XM_496446 2.038 LOC440886 XM_496572 2.150 LOC440995 XM498955 2.794 LOC441027 XM496707 4.039 LOC441164 XM_499041 -2.106 LOC494143 NM_001008708 -2.792 LOC51315 NM_016618 2.694 LOC55908 NM018687 -2.404 L0C56902 NM_020143 -2.008 LOC91461 NM_138370 -2.974 LOC92345 NM_138386 -2.410 LONPL NM_031490 2.205 LOX NM_002317 -3.558 LOXL2 NM_002318 5.544 LRIG3 NM_153377 -2.202 LRP 10 NM_014045 2.921 LSM11 NM173491 -2.254 LSM6 NM_007080 -3.351 LTB4DH NM_012212 2.193 LTBP3 NM 021070 2.269 LY96 NM015364 12.628 LYAR NM_017816 -2.678 MAC30 NM_014573 -2.204 MAD2L1 NM_002358 -2.509 MAK3 NM_025146 -2.015 MAL2 NM_052886 -2.739 MALATI -2.689 MAP 1 B NM_005909 /// NM_032010 2.450 MAP2K1IP1 NM021970 2.878 MAP3K8 NM_005204 2.425 MAPK6 NM_002748 -2.362 MAPKAPK5 NM_003668 /// NM_139078 -2.431 MARCH2 NM_001005415 /// NM_001005416 NM016496 2.223 MARCH8 NM001002265 /// NM001002266 /// NM_145021 2.143 MARCKS NM_002356 2.351 MARS2 NM_138395 -2.181 MASTL NM032844 -3.802 MATR3 NM018834 /// NM_199189 -2.259 MBL2 NM000242 -6.115 MBNL2 NM_144778 NM_207304 2.096 MBNL3 NM 018388 /// NM 133486 -2.263 MBTPS 1 NM_003791 /// NM201268 2.229 MCAM NM_006500 -2.701 MCM10 NM_018518 /// NM_182751 -3.796 MCM2 NM_004526 -2.365 MCM3 NM_002388 -2.442 MCM4 N1v1_005914 /// NM_182746 -3.179 MCM5 NM_006739 -2.670 MCM6 NM005915 -2.530 MCM7 NM_005916 /// NM182776 -2.518 MCM8 NM_032485 NM 182802 -2.431 MED6 NM005466 -2.903 MED8 NM001001651 /// NM_001001653 /// NM_001001654 /// -2.346 NM_052877 MEIS4 NR_002211 2.188 MELK NM_014791 -2.508 MESDCI NM_022566 -2.667 MET NM_000245 2.017 METRNL NM001004431 3.008 MGAT4A NM012214 -2.283 MGC11102 NM_032325 -2.793 MGC12916 --- -2.258 MGC13170 NM_199249 /// NM199250 -2.022 MGC13204 NM_031465 -3.680 MGC14289 NM_080660 -4.655 MGC23909 NM_174909 -3.516 MGC2408 NM_032331 -2.609 MGC24665 NM_152308 -2.169 MGC2560 NM_031452 -3.099 MGC26963 NM_152621 2.060 MGC34646 NM_173519 2.241 MGC4308 NM_032359 -2.688 MGC4399 NM_032315 -2.331 MICAL2 NM_014632 2.546 MICB NM005931 -3.377 MIXL1 NM_031944 -2.332 MK167 NM_002417 -2.093 MLF1IP NM_024629 -2.888 MLLT11 NM_006818 2.581 MMP3 NM_002422 6.834 MMP7 NM_002423 2.068 MNAB NM_018835 2.021 MNS 1 NM018365 -2.248 MOAPI NM_022151 3.702 MR-1 NM_015488 /// NM022572 -2.858 MRS2L NM_020662 -2.929 MSH6 NM000179 -2.485 MSLN NM_005823 /// NM_013404 2.215 MSRB3 NM_001031679 /// NM198080 2.183 MT1E NM_175617 2.113 MT1F NM_005949 2.261 MT1H NM 005951 2.084 MT1M NM_176870 2,212 MT 1 X NM_005952 2.354 MT2A NM005953 2.117 MTF2 NM 007358 -2.805 MTFRl NM_014637 -2.113 MTMR11 NM_006697 /// NM_181873 2.000 MUC 13 NM033049 2.314 MUC15 NM145650 3.095 MUTED NM_201280 -2.263 MVP NM_0051151// NM_017458 3.138 MXII NM001008541 /// NM_005962 /// NM_130439 2.208 MXRA7 NM_001008528 /Il NM_001008529 //I NM198530 2.162 MXRA8 NM_032348 2.884 MYBL 1 XM_034274 -2.095 MYCBP NM012333 -2.250 MYO I5B XM_496245 /I/ XR_000222 3.170 MYO 1 D NM 015194 2.547 MYO5A NM000259 2.215 MYO6 NM_004999 2.052 NABI NM_005966 -2.059 NAPILl NM_004537 /ll NM_139207 -2.445 NARG 1 NM_057175 -2.798 NASP NM_002482 IlI NM_1522981// NM_172164 -2.574 NBR2 NM_005821 NM_016632 -2.022 NCF2 NM_000433 2.827 NDRGI NM_006096 3.097 NDRG4 NM 020465 /// NM_022910 2.192 NEGR1 NM173808 2.987 NEIL3 NM_018248 -2.808 NEK2 NM 002497 -2.061 NEK3 NMv_002498 Il1 NM_152720 -3.046 NEXN NM144573 3.622 NFIB NM_005596 2.456 NIDI NM_002508 3.011 NID67 NM_032947 -3.881 NIPSNAP3A NM_015469 2.121 NKIRASI NM_020345 -3.233 NME6 NM 005793 -2.748 NMI NMy004688 2.343 NOL11 NMV015462 -2.162 NOL3 NM_003946 2.087 NOL5A NM 006392 -2.058 NOLCI NM004741 -2.586 NPC 1 L1 NM_013389 2.007 NR1D2 NM005126 3.752 NRIH4 NM_005123 -3.071 NR2FI NM 005654 2.131 NRAS NM002524 -2.563 NRBP2 NM_178564 2.311 NSF Il1 LOC641522 NM 006178 -2.505 NTN4 NM_021229 3.147 NU.FTP 1 NM_012345 -2.064 N[JP 160 NM_015231 -2.055 NUP205 NM_015135 -2.050 NUP35 NM001008544 /// NM_138285 -3.113 NUP37 NM_024057 -2.080 NUP50 NM_007172 /// NM_153645 lll NM153684 -2.083 NUP98 NM_005387 /// NM_016320 IlI NM139131/// NM139132 -3.648 NUPLl NM_001008564 /// NM_001008565 /// NM 014089 -2.031 NY-REN-41 NM_030771 /// NM_080654 -2.489 NY-SAR-48 NM_001011699 /// NM 033417 -2.002 OAS1 NM001032409 /Il NM002534 !1I NM_016816 2.024 OPTN NM_001008211 11/ NM_001008212 /// NM_001008213 /l/ 2.192 ORC 1 L NM_004153 -2.644 ORC6L NM_014321 -2.268 ORMI NM 000607 -3.646 ORMI /!/ ORM2 NM_000607 NM_000608 -3.184 ORM2 NM_000608 -3.528 OSTheta NM178859 -2.181 OSTMI NM_014028 2.162 P8 NM012385 3.789 PA2G4 NM_006191 -2.761 PABPC4 NM_003819 -2.669 PACS2 NM015197 2.049 PAICS NM_006452 -2.288 PAKiIP1 NM017906 -2.110 PANXI NM_015368 2.031 PAPSS2 NM_001015880 /// NM_004670 2.144 PAQR5 NM_017705 2.302 PARD6B NM_032521 -2.381 PARP 11 NM_020367 2.069 PAX6 NM000280 /// NM_001604 2.439 PBK NM_018492 -2.683 PCAF NM_003884 3.169 PCLKC NM017675 2.991 PCTP NM021213 -3.039 PCYTIB NM_004845 -2.007 PDGFA NM_002607 /// NM_033023 2.105 PDGFC NM_016205 2.068 PEG3 NM 006210 -3.673 Pfs2 NM016095 -3.969 PGCP NM_016134 2.061 PGRMCI NM_006667 -2.576 PHF19 NM_001009936 /// NM_015651 -2.739 PHP20L1 NM_016018 /// NM_024878 Ill NM_0322051/I NM_198513 2.616 PHLDAI NM_007350 5.217 PHLDB3 NM_198850 2.219 PIGA NM_002641 /// NM_020472 /// NM 020473 -3.778 PIGC NM_002642 /// NM_153747 -2.005 PIGL NM004278 -2.091 PTNK1 NM 032409 2.015 PIP5KIB NM 001031687 /// NM 003558 -3.370 PITPNCI NM_012417 /ll NM_181671 2.003 PJA2 NM_014819 2.727 PKNOXI NM_004571 lll NM_197976 2.032 PLAGL1 NM_002656 /// NM_006718 -2.210 PLAGL2 NM 002657 -5.050 PLAU NM002658 2.556 PLEKHA2 XM_496973 2.152 PLEKHH2 NM_172069 2.260 PLEKHMI NM_014798 2.350 PLKI NM_005030 -2.144 PLK4 NM014264 -2.560 PLXNB2 XM_371474 2.041 PNN NM_002687 -2.282 PNRC 1 NM_006813 2.333 POLA NM_016937 -2.150 POLE2 NM_002692 -3.902 POLRIB NM_019014 -2.388 POLR2D NM004805 -2.627 POLR3G NM006467 -3.493 POLR3K NM_016310 -2.120 POPDC3 NM022361 2.240 PPAT NM_002703 -2.504 PPIH NM006347 -2.170 PPIL5 NM_152329 /1/ NM_203466 lll NM_203467 -2.440 PPP1R13B NM_015316 -2.742 PPP4C NM_002720 -2.176 PQLC3 NM152391 3.083 PRAFI NM_022490 -2.021 PRAP 1 NM_145202 2.151 PRIM 1 NM_000946 -2.588 PRIM2A NM_000947 -2.124 PRKAR2A NM_004157 -2.618 PRKCA NM_002737 2.135 PROCR NM006404 2.102 PRTG XM_370866 -6.751 PSFI NM_021067 -3.393 PSME4 NM_014614 -3.866 PTP4A1 NM_003463 2.246 PTPRM NM_002845 2.376 PTPRN2 NM_002847 IlI NM_130842 lll NM_130843 2.309 PTXI NM_016570 -2.405 PUNC NM_004884 -2.713 PURB NM_033224 2.249 PYCARD NM_013258 IlI NM_145 1 82 /1/ NM_145183 2.306 QKI NM_006775 lll NM_206853 /1/ NM_206854 Il1 NM_206855 2.695 RAB11PIP4 NM_032932 -2.066 RAB31 NM_006868 2.585 RABEP2 NM_024816 2.771 RABGGTB NM_004582 -2.177 RAD18 NM_020165 -4.207 RAD51 NM_0028751// NM_133487 -2.850 RAD51AP1 NM 006479 -2.986 RALGDS NM006266 2.134 RANBPl NM_002882 -2.161 RAP2B NM_002886 2.205 RASD 1 NM_016084 5.105 RASSF2 NM_014737 /// NM_170773 /// NM_170774 2.947 RBBP7 NM_002893 -2.295 RBM14 NM_006328 -2.481 RBM19 NM_016196 -2.041 RBM24 NM_153020 3.762 RBP 1 NM_002899 2.370 RBPMS NM_001008710 /// NM 001008711 NM 001008712 /// -2.087 RECK NM_021111 2.950 RFC2 NM_002914 /// NM_181471 -2.300 RFC3 NM 002915 /// NM_181558 -3.259 RFC4 NM002916 /// NM_181573 -2.337 RFC5 NM_007370 11/ NM_181578 -3.462 RFFL NM_001017368 /// NM_057178 -2.044 RFWD3 NM_018124 -3.699 RGS3 NM_017790 /// NM_02 1 106 /// NM_130795 /// NM_134427 -2.786 RHOB NM_004040 -2.149 RHOQ NM_012249 2.563 RHOQ NM_012249 /// XM209429 3.585 RIF 1 N1VI_018151 -2.269 RIMS3 NM_014747 2.204 RIPK5 NM_015375 /// NM_199462 2.354 RITI NM006912 2.081 RNF144 NM_014746 2.113 RNU22 NR_000008 -2.920 RNU47 XR000223 -2.614 RPS6 NM_001010 -2.315 RPS6KA3 NM_004586 -2.009 RPUSD3 NM_173659 -3.293 RRAGD NM_021244 2.188 RRMl NM_001033 -2.328 RRM2 NM001034 -4.193 RRM2B NM_015713 2.704 RRN3 NM_018427 -2.007 RSC 1A1 NM_006511 -3.230 RTCD 1 NM 003729 -2.223 RTF 1 NM_015138 2.048 RTN2 NM_005619 NM_206900 /// NM_206901 NM206902 2.095 RTN4IP1 NM_032730 -2.407 RYl NM006857 2.180 S100A2 NM 005978 5.992 S100A4 NM002961 ///NM019554 2.395 S 100A6 NM_014624 3.585 S100PBPR NM_001017406 ///NM_022753 -2.885 SACS NM 014363 -2.165 SAR1B NM_001033503 /// NM_016103 2.287 SASS6 NM 194292 -2.493 SAT NM_002970 2.290 SCAMPI NM_004866 /// NM_052822 2.098 SCARB2 NM_005506 2.032 SCD NM_005063 -2.328 SCGN NM_006998 -2.461 SCN9A NM_002977 3.362 SCPEP I NM021626 2.260 SELM NM_080430 2.480 SEMA3B NM_001005914 /// NM004636 2.142 SEMA3G NM_020163 2.055 SEPT6 /// N-PAC NM_015129 /// NM 032569 /// NM 145799 /// NM 145800 2.143 SERP 1 NM_014445 -2.007 SERPINA3 NM_001085 2.456 SERPINA6 NM001756 -2.066 SERPINE1 NM_000602 2.400 SEZ6L2 NM_012410 /// NM_201575 2.116 SFRS 1 NM_006924 -2.031 SGCB NM_000232 2.304 SGK3 NM_001033578 /// NM_013257 /// NM_170709 -2.097 SGOL2 NM_152524 -2.263 SH3BGRL NM_003022 2.010 SH3BGRL3 NM031286 2.340 SH3BP5 NM_001018009 /// NM_004844 2.097 SH3GLB 1 NM_016009 2.256 SHCBPI NM_024745 -2.480 SIL NM_003035 -2.173 SIP I NM_001009182 /// NM_001009183 NM_003616 -2.194 SKP2 NM_005983 /// NM_032637 -4.277 SLC16A10 NM_018593 -2.944 SLC16A6 NM_004694 2.408 SLC17A2 NM_005835 -2.411 SLC20A1 NM_005415 -2.298 SLC22A18 NM_002555 /// NM_183233 2.717 SLC22A7 NM_006672 /// NM_153320 -2.377 SLC23A2 NM_005116 /// NM_203327 2.410 SLC25A13 NM_014251 -2.585 SLC25A24 NM_013386 /// NM_213651 -2.124 SLC25A32 NM_030780 -2.835 SLC26A11 NM_173626 2.537 SLC2A3 NM_006931 -6.221 SLC2A3 NM_006931 /// NM_153449 -5.017 SLC2A8 NM_014580 -2.078 SLC30A10 NM 001004433 /// NM_018713 -2.129 SLC35D2 NM_007001 -2.343 SLC35F5 NM_025181 -3.794 SLC38A5 NM_033518 -2.093 SLC39A14 NM_015359 -3.916 SLC40A1 NM_014585 5.218 SLC43A1 NM_003627 -2.391 ST,C44A1 NM_022109 /// NM_080546 2.114 SLC44A5 NM_152697 2.821 SLC4AII NM_032034 4.907 SLC4A5 NM_02 1 196 /// NM 033323 /// NM_133478 /// NM_133479 -2.069 SLC5A6 NM_021095 -2.583 SLC6A14 NM007231 -2.725 SLC6A6 NM_003043 2.081 SLC7A11 NM014331 2.056 SLC7A2 NM_001008539 !// NM003046 2.115 SLC7A6 NM_003983 -2.170 SLCO4C1 NM_180991 -6.128 SMAD2 NM 001003652 /// NM_005901 2.496 SMARCA2 NM003070NM_139045 2.328 SMARCCI NM_003074 -2.014 SMC1L1 NM_006306 -2.248 SMC2L1 NM_006444 -2.288 SMPDI NM_000543 NM_001007593 2.164 SMURF2 NM_022739 2.381 SNAP23 NM_003825 /// NM_130798 -2.346 SNAPC5 NM_006049 -2.093 SNX5 NM_014426 NM 152227 -2.669 SOAT2 NM_003578 -2.669 SOCS 1 NM003745 -2.760 SOLH NM_005632 -2.134 SOX4 NM003107 2.011 SPBC25 NM_020675 -2.506 SPCS3 NM_021928 -3.408 SPIN2 !// SPIN-2 NM_001006681 /// NM_001006682 lll NM_001006683 2.031 SPON2 NM_012445 4.946 SPTANI NM_003127 2.050 SPTBNI NM 003128 (/( NM 178313 2.029 SPTLC2L --- -2.194 SQSTMI NM003900 2.525 SR140 XM_031553 -2.333 SSX2IP NM_014021 -2.558 ST6GALNAC2 NM_006456 -3.504 STEAP3 NM_001008410 /// NM_018234 NM_182915 2.363 STK17A NM_004760 2.089 STK40 NM032017 -2.417 STK6 NM_003600NM_198433 /// NM_198434 NM_198435 /// -4.188 STS-1 NM_032873 -3.120 STX3A NM 004177 -2.978 SULTICI NM_001056 Ill NM_176825 2.091 SUPT16H NM_007192 -2.214 SUSD2 NM_019601 3.786 SUV39H2 NM_024670 -3.885 SYNGR3 NM_004209 2.892 SYTLI NM_032872 2.936 SYTL2 NM_032379 NM_032943 NM_206927 NM_2069284.644 TACC2 NM_006997 /// NM_206860 NM_206861 NM_206862 2.436 ITACC3 NM 006342 -2.037 TAF5 NM006951 -3.117 TAF5L NM001025247 /// NM014409 -2.378 TAGLN NM001001522 /// NM_003186 2.095 TBC1D3 NM_001001418 /1/NM_032258 3.067 TBRG4 NM004749 NM_030900 /// NM_199122 -2.044 TBX3 NM005996 NM016569 2.237 TCERG 1 NM_006706 -2.015 TCOF1 NM000356 /!/ NM001008656 /// NM_001008657 -2.455 TCTEIL NM_006520 2.633 TDE2L NM_178865 3.110 TDP I NM_00 1 008744 /// NM_018319 -2.141 TEAD4 NM003213 /// NM_201441 /// NM201443 -2.730 TEPI NM007110 2.202 TFAM NM_003201 -2.004 TFDP1 NM_007111 -2.046 TFRC NM_003234 -2.504 TGFA NM003236 2.034 TGFB 1 NM_000660 2.104 TGFB 1I 1 NM_015927 2.701 TGFBR3 NM_003243 -3.258 THEM4 NM_053055 /// NM_176853 -2.356 TIMM8A NM_004085 -2.159 TIMP2 NM003255 2,818 TK1 NM003258 -2.182 TK2 NM_004614 3.123 TM4SF5 NM_003963 2.404 TMCO3 NM_017905 2.169 TMEFFI NM_003692 2.028 TMEMI6K NM018075 2.391 TMEM48 NM_018087 -2.540 TMEM55A NM018710 2,227 TMEM57 NM018202 /// NM145284 2.295 //! LOC159090 TMEM8 NM021259 -2.039 TMEM87B NM_032824 2.540 TMPO NM001032283 /// NM_001032284 /// NM_003276 -2.608 TMSB4X /// TMSL3 NM_021109 /// NM183049 2.137 TncRNA --- 2.342 TNFRSFIIA NM_003839 2.156 TNNFRSF14 NM_003820 2.220 TNFSFIO NM_003810 -2.099 TNRC6A NM_014494 /// NM020847 2.038 TNRC8 --- 2.767 TOPIMT NM_052963 2.350 TOP2A NM_001067 -2.202 TOPBP 1 NM007027 -2.021 TP5313 NM_004881 /// NM147184 2.473 TP531NP1 NM 033285 2.382 TPBG NM006670 3.351 TPM2 NM_003289 /// NM213674 3.855 TPR NM_003292 -2.385 TPX2 NM 012112 -2.044 WO 2008/073922 PCT(1JS20071087037 TRAF5 NM001033910 /// NM_004619 NM_145759 2.173 TRIB2 NM_021643 2.122 TRIM2 NM_015271 3.066 TRIM22 NM_006074 3.115 TRIM24 NM_003852 ///NM_015905 2.249 TRIM56 NM_030961 2.327 TRIP13 NM_004237 -2.384 TRPV2 NM_016113 2.038 TSC22DI NM_006022 /// NM_183422 2.076 TTC3 NM_001001894 /// NM_003316 2.040 TTC7B NM_001010854 2.297 TTK NM003318 -2.295 TTLIA NM014640 -4.693 T1'YH2 NM_032646 /// NM_052869 2.176 TUBA3 NM_006009 6.172 TUBB2 NM_001069 2.343 TUBB-PARALOG NM178012 3.758 TUBEI NM_016262 -2.325 TUBG 1 NM_001070 -2.279 TUSC2 NM007275 -2.216 TUSC3 NM_006765 /// NM_178234 2.119 UBE2H NM_003344 /// NM_182697 2.381 UBE2Q2 NM_173469 2.207 UBE2T NM_014176 -2.756 UCHL5 NM_015984 -2.974 UGCG NM_003358 -2.081 UHMKI NM 175866 2.111 UHRF1 NM_013282 -4.543 UIPI NM017518 NM_207106 /// NM_207107 -2.016 ULKI NM_003565 2.003 UNC93A NM 018974 -2.304 USPIO NM_005153 -2.312 UTP 15 Nlv1032175 -2.352 VAMP i NM_014231 /// NM_016830 /// NM199245 2.079 VAMP3 NM004781 -2.043 VLDLR NM_001018056/// NM_003383 2.084 VNN1 NM_004666 2.180 VPS33A NM022916 -2.148 VPS54 NM_001005739 //1 NM_016516 -2.421 VRK1 NM_003384 -3.224 WBPII NM_016312 -2.777 WDHD i NM001008396 /// NM_007086 -3.432 WDR45 NM_001029896 /// NM_007075 2.493 WIG1 NM_022470 /1/NM_152240 2.413 WNK4 NM_032387 2.064 XPO4 NM_022459 -3.104 XPO5 NM_020750 -2.248 YIPF4 NM_032312 2.158 YOD 1 NM_018566 -3.320 YPEL3 NM031477 3.060 YPEL5 NM_016061 3.006 YWHAH NM 003405 2.764 ZA20133 NM_019006 2.086 ZBTB20 NM015642 2.163 ZBTB4 NM_020899 2.731 ZCCHCIO NM_017665 -2.061 ZCCHC9 NM_032280 -3.491 ZCSL2 NM_206831 -4.066 ZDHHC2 NM016353 2.520 ZFP90 1`IM_133458 2.099 ZHX3 NM015035 2.008 ZNF 117 NM024498 2.157 ZNF161 NM_007146 2.163 ZNF200 NTM_003454 11/ NM_198087 NM_198088 -2.419 ZNF226 NM_001032372 /// NM 001032373 /// NM_001032374 /// 3.068 NM 001032375 /// NM^015919 ZNF267 NM_003414 -2.131 ZNF329 NM_024620 2.017 ZNF432 NM_014650 2.618 ZNF514 N141_032788 2.073 ZI+IF678 NM 178549 -2.169 ZNF680 NM178558 2.339 ZNF689 NM138447 -2.188 ZNF706 NM_016096 2.074 ZNF708 NM_021269 2.382 ZNF83 NM_018300 2.269 ZRF1 XM_168590 /// XM_379909 -2.004 ZWILCH NM 017975 -3.135 ZWINT NM001005413 /// NM_00 1005414 /// NM_007057 !1/ -2.272 NM_032997 ZYX NM 001010972 /!/ NM 003461 2.039 Table 4. Genes with altered mRNA expression levels in HL-60 cells, following transfection with pre-miR hsa-let-7b.
RefSeq Gene Fold TranscriptID
Symbol (Pruitt et al., 2005) Change AATF NM_012138 -2.27 AB020674, AB020674, AF245481 -2.89 AB032979 AB032979 -2.41 AB033091, pB033091, NM_020342 -3.26 AB058774 AB058774 2.43 AB062477 AB062477 3.08 AB083483 AB083483 3.71 ABCA3 BC062779, NM 001089 2.05 ABCF2 NM 005692 -2.88 ACADVL BC020218, NM 000018 -2.65 ACP1 NM 004300, NM_007099, NM 177554 -2.71 ADIPOR2 NM 024551 -2.64 AF011390 AF011390 2.32 AF090928 AF090928 -3.71 AF116680 AF116680 2.75 AF240698 AF240698 -2.14 AF277180 AF277180 -2.27 AF289562 AF289562 2.29 AF289565 AF289565 2.55 AF346307 AF346307 2.55 AF439711 AF439711 2.73 AF445026 AF445026 2.17 AF502589 AF502589 -2.11 AHCY M61831, NM 000687 -3.55 AJ515384 AJ515384 -3.09 AK001073, Ag001073, BC080641 2.72 AK001987 AK001987 2.28 AK001998 AK001998 2.31 AK022118 AK022118 2.54 AK024110 AK024110 2.08 AK024190 AK024190 -2.94 AK026367 AK026367 2.11 AK026780 AK026780 2.36 AK027395, AL136861, AY358413, AK027395, AL136861, AY358413, BC063012, CR593410 2.65 BC063012, AK027583 AK027583 2.06 AK054654 AK054654 2.7 AK054935, qg054935, NM 152479 2.27 AK056176 AK056176 2.7 AK057017 AK057017 2.65 AK057222, AK057222, NM 145038 3.2 AK057372 AK057372 2.23 AK058196 AK058196 2.19 AK090733, AK090733, BC003505 2.92 AK091523 AK091523 2.63 AK093431 AK093431 2.17 AK094354 AK094354 3.32 AK095939, AX095939, BC003083 -2.39 AK096571 AK096571 2.1 AK097091, AK097411, AK097091, AK097411, NM_207331 4.51 AK097411, BC050737, AK097411, BC050737, NM_207331 4.51 AK123855, AK123855, BC006300 2.89 AK124968 AK124968 -2.22 AK125351 AK125351 2.67 AK125522, A{125522, NM 004691 -2.35 AK125850 AK125850 -2.91 AK125850, AK125850, AL833349 -2.91 AK126051 AK126051 2.47 AK126465 AK126465 -2.88 AK127284 AK127284 -2.03 AK127639 AK127639 2.17 AK127692, AK127692, NM_175614 -4.14 NDUFAII
AK128554 AK128554 -2.12 AK131383 AK131383 -2.3 AK131517, AK131517, BC063666 2.62 AK2 NM 013411 -3.48 AKAP5 NM 004857 4.41 AKAP8L NM 014371 -2.23 AKRICL2 AB040821, AB040822, AF263242, NM_031436 2.93 ALDH3A2 NM000382 3.22 ALG 1 NM 019109 -2.31 ALOX15B AF468053 3.81 ALOX5AP NM_001629 -2.86 ALS2CRI9 AB073472, AF428250, AF428251, AF466152, NM152526, _3.48 ALS2CR7 NM 139158 3.02 AMDi BC000171 -2.78 ANP32C NM_012403 2.65 ANTXRI A.K001463, NM 053034 3.75 ANXA6 AK130077, NM001155, NM_004033 -2.37 APBA3 NM 004886 2.01 APG3L NM 022488 -3.43 APLP2 BC000373 -2.81 ARHGAP18 AL834511,NM_033515 3.11 ARHGAP26 BC068555, NM015071 2.01 ARIDIA AF231056, AF268913, AF521670, NM_006015, NM_018450, -3.37 ARPC5, BC057237, BC057237, BC071857, NM_005717 -2.51 ASFIB NM 018154 -2.61 ASNAI NM 004317 -2.03 ATF3 AB078026, AY313926, AY313927 -3.8 ATF4 NM 001675 -2.09 ATP2A2 NM 001681, NM 170665 -4.57 ATP5G3 NM 001002256 -2.27 ATP6VIF NM 004231 -2.76 ATRX NM 000489, NM 138270, NM 138271, U72937 2.1 ATXN7L2 BC036849 3.95 AY081145 AY081145 -2.05 AY099328, AY099328, BC002509 2.53 AY345239, AY345239, NM 024773 -2.5 AY358738 AY358738 4.71 AY692447, BC040622, AY692447, BC040622, NM182761 -2.93 AYP 1 NM 032193 3.72 BAG1 AF116273 -2.94 BAG2 NM 004282 -2.84 BAG5 NM 001015049, NM 004873 -2.24 BANFI NM 003860 -2.92 BAT2 NM 004638, NM 080686 -2.53 BC004492 BC004492 2.02 BC006177 BC006177 -4.17 BC007516 BC007516 4.53 BC009792 BC009792 3.08 BC011671 BC011671 2.63 BC013796 BC013796 -2.96 BC014654 BC014654 -2.39 BC016050 BC016050 -2.4 BC016654 BC016654 -2.16 BC020256 BC020256 -2.36 BC020670 BC020670 2.37 BC021187 BC021187 -2.62 BC025700 BC025700 -3.61 BC029496 BC029496 2.3 BC029580 BC029580 3.49 BC030200 BC030200 2.64 BC032334 BC032334 -2.39 BC032396, BC032396, BC041379 2.14 BC032420 BC032420 2.74 BC035554 BC035554 -3.11 BC035875 BC035875 -2.74 BC035935, BC056271, BC035935, BC056271, NM_016627 -3.34 BC036832 BC036832 -4.43 BC040013 BC040013 4.24 BC040441, BC040441, BC068599 2.12 BC041860, BC047720, BC041860, BC047720, BX647229 2.25 BC045618, BC045618, BC057784 -3.92 BC062325 BC062325 -4=02 BC064430 BC064430 -3.1 BC064479 BC064479 2.05 BC065557 BC065557 2.03 BC066124, BC066124, BC066775 -2.43 BC066644 BC066644 -2.35 BC073829 BC073829 -2.5 BC093044 BC093044 -3.07 BEXL1 BC015794 -2.54 BFAR NM016561 -2.77 BINI AF068916, NM_139346, NM 139348 2.42 BIN2 BC047686, NM 016293 4.57 BMP2 NM_001200 -5.22 BPI BC032230, NM 001725 2.96 BRAP NM006768 -2.09 BST2 AK223124, NM004335 -2.06 BZRP NM 000714, NM 007311 -3.32 ClOorf45 BC064407, NM 031453 -2.48 ClOorf67 BC035732, NM 153714 2.26 ClOorf94 BC034821 -2.45 C12orf12 NM 152638 3.91 C14orf103 NM 018036 2.12 C14orfl53 NM 032374 -2.55 C14orf48 AK097741, NM152777 -3.86 C15orf12 NM 018285 -3.2 C17orf27 BC032220, BX647946, NM 020914 -2.41 C19orf25 BC018441, NM_152482 3.8 Clorf26 BC030781, NM 017673 2.98 Clorf64 NM178840 2.08 C1QBP NM 001212 -2.72 CIQTNF2 NM031908 2.36 C 1 QTNF3 NM_030945, NM 181435 2.13 C20orf27 BC024036, CR615129, NM 017874 4.34 C2orf29 NM 017546 -2.92 C3orfl0 NM 018462 -3 C4orf16 BC009485, BX647702, NM018569 -2.7 C5orf19 AK223611, NM_016606 3.03 C6orflO8 NM 006443 -2.49 C6orf128 BC026012, BC029657, NM_145316 3.24 C6orf136 BC073975, NM 145029 -2.41 C6orF155 NM 024882 -2.56 C6orf62 NM_030939 -2.58 C6orf69 AY305862, BC023525, NM 173562 -4.01 C6orf96 AK000634, NM_017909 -2.69 C8orf6 AJ307469 -2.59 C9orfl56 BC002863, NM 016481 2.66 C9orfl6 NM 024112 -2.69 C9orf46 NM 018465 3.54 CABP7 NM 182527 -2.56 CACNAIA AB035727 2.21 CACNA2D3 AF516696, AJ272213, NM 018398 2.23 CAD NM 004341 -3.71 CAMKID NM 020397, NM 153498 2.07 CAMK2N1 NM 018584 2.1 CAPNSI BC011903,NM_001749 -2.89 CASC3 BC044656 3.03 CASP6 NM 001226 -2.27 CAST NM 015576 2.29 CBXI NM 006807 -2.72 CCNDBPI AK075146, AK128849, BC009689, NM 012142, NM 037370 -2.62 CCT4 NM 006430 -2.77 CCT7 NM 006429 -2.92 CD81 NM_004356 -2.48 AB209095, AF067519, AF067520, AF067521, AF067522, AF067523, AF067525, NM_001787, NM_024011, NM033486, CDC2LI NM_033488, NM_033489, NM_033490, NM__033492, -218 NM_033493, NM 033528, NIVI 033529, NM_033531, NM_033534, NM_033537, U04816, U04817, U04818, U04824, AB209095, AF067512, AF067514, AF067516, AF067520, CDC2L2 AF067521, AF067522, AF067523, AF067525, NM_033534, -2.18 NM 033536, NM 033537 CDC45L AJ223728, CR604288, NM 003504 -3.47 CDKL1 NM 004196 2.35 CEACAM6 BC005008, M18728, NM 002483 3.85 CEBPE NM 001805 -3.42 CGI-128 NM 016062 -3.04 CGI-63 BC001419, NM_016011 -3.3 CHAD NM 001267 -4.68 CHCHDI NM 203298 -2.64 CHMP2A NM 198426 -2.93 CHRNBI NM_000747 2.12 CHST3 NM 004273 2.23 CINP NM 032630 -2.09 CIP29 NM 033082 -2.9 CLC NiVi 001828 2.59 CLICI NM_001288, X87689 -3.8 CLTA NM 001833 -3.71 CMAS BC016609, NM 018686 -2.37 CNTN4 NM 175607, NM 175612 2.23 CNTNAP3 AK054645, NM 033655 2.31 COLIA2 NM 000089 2.35 COPA BC038447, NM 004371 -3.21 COQ3 CR607786, NM 017421 -2.3 COROIA AB209221, NM 007074 -4.94 COTL1 AK127352, NM 021149 -4.4 COXBA NM 004074 -2.48 CPNE1 NM 152930 -2.68 CR602867 CR602867 -4.41 CR605850 CR605850 -4.54 CR607440 CR607440 2.35 CR933646 CR933646 -2.56 CRHBP NM 001882 2.31 CRR9 AK126225, BC025305, NM 030782 -2.85 CSFl NM 000757, NM 172211 2.57 CSMD2 AK122603, AK127722 -3.29 CSNK2B CR592250, NM 001320 -2.73 CTNS BC032850, NM 004937 2.58 CULl BC034318, NM003592, U58087 -2.34 CUL7 NM 014780 4.34 CXorf9 NM 018990 -2.24 CXXC1 BC015733, NM_014593 -2.94 CYC 1 NM 001916 -3.41 CYP2B6 NM 000767 -2.22 DDOST D29643 -2.41 DDX39 BC032128, NM 005804, NM_138998 -4.77 DDX50 NM 024045 2.67 DGCR6 BC047039, NM 005675 -2.45 D14X30 BC038417, NM_014966, NM138614, NM_138615 -2.61 DHX35 AK025541, BC033453, NM 021931 -2.09 DISPI AK056569, NM032890 2.42 dJ39G22.2 NM_001008740 2.91 DKFZp451JO BC046565, NM 175852 -2.88 DKFZP564JO NM 015459 -2.77 863 _ DNAJC10 AF314529, AK027647, AL832632, AY089971, AY358577, 2 32 BC034713,NM 018981 DNAJC12 NM 201262 -2.07 DNCLII NM016141 -2.56 DNCLI2 NM 006141 4.94 DNM2 AK097875, AK124881, NM 001005360, NM001005361, 2.98 NM_001005362, NM004945 DONSON NM_017613, NM145794, NM_145795 -2.13 DRD3 L20469 -2.47 DRG1 NM004147 -2.5 DVLI AK093189, NM_004421, NM_181870, NM_182779, U46461 4.92 EBF2 AY700779, NM_022659 2.13 EBPL BC021021, BC073152, NM_032565 -3.09 EDIL3 BC053656, NM005711 2.06 AF267861, BC019669, BC071619, BC094687, CR598396, EEF1A1 CR623309, NM 001402 -2.82 EEF1D NM001960, NM_032378 -3.63 EEFIG AF119850, NM_001404 -2.67 EEF2 NM001961 -3.58 EIF2S3 BC019906 -3.08 EIF3S4 NM_003755 -2.83 EIF3S8 BC001571, NM 003752 -3.92 EIF4A1 NM_001416 -4.52 EIF4EBP1 NM 004095 -2.42 ELF1 BC030507, NM172373 -3.56 EMP3 NM001425 -3.12 ENOI BC073991, NM 001428 -6.06 ENO3 NM001976 2.02 EPB41 BC039079 4.22 EPM2A AF454493, NM 005670 2.91 FAM50A CR612868, D83260, NM_004699 -2.26 FAM54B AF173891, AK056721, BC017175, NM 019557 -3.41 FASN BC063242, NM 004104 -3.02 FBL NM001436 -4.61 FBP2 NM003837 2.01 FBXO17 AK021860, NM 024907, NM 148169 2.09 FBX040 AB033021,NM 016298 2.78 FBXO42 NM018994 2.73 FH NM 000143 -2.05 FIBP NM~_004214, NM 198897 -3.44 FLJ10006 AK056881, BC017012, NM 017969 3.46 FLJ10490 NM018111 2.01 FLJ10774 NM024662 -3.89 FLJ11305 NM018386 -2.45 FLJ12760 NM 001005372 -2.8 FLJ14816 NM032845 -2.2 FLJ20641 BC050696, NM_017915 3.04 FLJ23322 BC027716, NM 024955 -2.05 FLJ25143 NM 182500 -2.54 FLJ25471 AK058200, NM_144651 -2.19 FLJ31139 BC064898, NM_173657 2.1 FLJ35740 NM147195 -2.07 FLJ36070 AK131427, NM_182574 -2.37 FLJ36180 BC015684, NM 178556 2.54 FLJ37794 NM 173588 -3.02 FLJ42461 NM 198501 -5.18 FLJ90650 BC094716, NM 173800 2.32 FOXP3 NM 014009 -4.42 FPGS BC009901, BC064393, M98045, NM_004957 -2.9 FSCNI NM 003088 3.86 FTL BC067772 -5.74 FTSJI NM 012280, NM 177439 -2.97 FUT8 NM 004480, NM 178155, NM 178157 2.07 FXYD5 AF177940, NM 144779 -2.56 G1P3 NM 002038, NM 022872, NM 022873 4.05 GAA NM 000152 2.09 GABARAP NM 007278 -2.82 GABRB2 NM 000813, NM_021911 2.42 GANAB BC065266, NM 198334, NM_198335 -2.08 GAPDH NM 002046, X53778 -3.7 GBA2 AB046825, AK057610, NM 020944 -2.44 GBL AK021536, AK022227, BC052292, NM 022372 -2.59 GBP3 BC063819, CR936755, NM 018284 2.75 GCHFR NM 005258 -2.56 GDI2 NM 001494 -3.77 GH1 AF185611 -2.17 GIP NM 004123 2.16 GLT25D1 AK075541, BC020492, NM 024656 -2.99 GLUD2 BC050732, NM 012084 -2.12 GMDS AF040260, BC000117, NM 001500 -2.83 GNB2 NM 005273 -3.1 GNB2LI AY336089, CR609042, NM 006098 -2.34 GOR NM 172239 2.04 GOR NM 172239 2.04 GOR NM 172239 2.04 GPR BC067106, NM_007223 2.9 GPR18 NM 005292 2.11 GPR3 NM 005281 -2.04 GPSN2 CR593648, NM 004868, NM 138501 -2.48 GPX1 BC070258, NM_000581 -4.59 GPX4 BC039849, NM_002085 -3.77 GRN AK023348, NM 002087 -2.7 GSTP1 NM 000852 -2.4 GTPBP4 NM 012341 -2.25 GUCYlA2 NM 000855, Z50053 2.45 GUKI AK125698, NM 000858 -3.31 GZMB AY232654, AY232656, AY372494, NM 004131 2.56 H1F0 CR456502 -3.21 HAND2 NM 021973 2.07 HAXI NM_006118 -2.45 HDAC7A AK024469, AK026767, AY302468, BC064840, NM015401, 5.6 HHAT BC051191, CR936628, NM 018194 2.19 HIST1H2BN BC009783, BC011372, NM 003520 2.1 HISTIH3G NM 003534 -2.62 HISTIH4C NM 003542 -2.19 HLA-E NM 005516 -2.12 HLCS NM 000411 2.08 HMG2LI NM 001003681, NM 005487 -2.97 HMGA1 BC071863, NM 002131, NM 145899 -2.37 HMGB1 NM 002128 -3.34 HNRPC BC003394, BC089438, BX247961, CR617382 -2.41 HNRPF BC016736, NM_004966 -2.88 HNRPL BC069184, NM 001533 -4.23 HRMTIL2 AY775289, NM 001536, NM 198318, NM 198319 -4.74 HS3STI NM 005114 -2.09 HSDL2 BC004331, NM 032303 -2.02 HSPA5 NM 005347 -4.78 HSPA8 BC016179, NM 006597, NM 153201 -3.45 HSPB2 NM 001541 -2.4 HSPCO23 NM 014047 -3.13 HSPCB AF275719, BC012807, NM 007355 -3.64 HSPDI BC002676, CR619688, NM 002156 -3.13 HYPC AK123353, BC067364, NM 012272 2.15 ICT1 NM 001545 -3.28 IER2 NM 004907 -2.68 IFIT5 BC025786 2.13 IFRD2 NM_006764, Y12395 -2.45 IGLV6-57 BC023973 4.93 IL22 NM 020525 2.23 IL6ST AB 102799, NM002184, NM 175767 2.3 ILDRI AY134857, AY672837, NM 175924 5.04 ILF2 NM 004515 -3.78 ILF3 AJ271747, NM 012218 -2.69 IN080 NM 017553 3.37 ITGA8 NM 003638 2.13 ITGB4BP NM 181466, NM 181468 -2.33 ITIH 1 NM 002215 -2.69 JUNB NM 002229 4.8 KIAA0082 NM_015050 -2.01 K1AA0284 BC047913, NM 015005 3.61 KIAA0339 NM 014712 3.32 KIAA1393 BC063551,NM 020810 -3.34 KIAA1533 AK074914, BC014077, NM_020895 -2.27 KIF20A NM 005733 3.95 KIF9 NM 022342, NM 182902, NM_182903 2.77 KIR2DL1 BC069344, NM 014218 2.82 KRTAP19-1 NM 181607 2.15 KRTAP4-2 NM 033062 2.78 LCP1 AK223305, NM 002298 -2.75 LENEP NM 018655 3.51 LETMDI AK127540, AY259835, AY259836, BC064943, NM 015416 -2.46 LFNG NM 002304 2.39 LGALSI NM 002305 -3.19 LGI4 BC087848 2.17 LMNBI Nlvl 005573 -2.69 LOC124402 AF447881,NM 145253 2.57 LOC129607 NM 207315 2.63 LOC152831 NM 175737 2.15 LOC153561 NM_207331 4.51 LOC157697 NM_207332 2.69 LOC220686, NM_199283, NM 199345 -3.45 LOC284001 NM 198082 2.7 LOC388389 NM213607 2.52 LOC388882 NM 001006606 -2.64 LOC440503 NM 001013706 2.42 LOC51149 BC069051, NM 001017987, NM 016175 2.2 LOC51233 AL080197, NM 016449 2.94 LOC51234 BC016348, NM 016454 -3.3 LRAT NM 004744 2.28 LRFN4 NM 024036 -2.09 LRP 12 NM 013437 2.13 LRP6 NM 002336 -2.39 LSM2 NM 021177 -2.41 LSM4 NM 012321 -2.63 LSP1 AK129684, NM 001013254, NM 002339 2.51 LY6G6D AF195764, NM_021246 4.62 AF244129, AK128573, AY007142, BC027920, BC062589, LY9 BC064485, IA2621, NM 002348 2'88 M6PRBPI AK223054, BC019278, NM_005817 -2.58 MAGEA2 NM 175743 -3.34 MAN1C1 AF'318353, NM 020379 2.69 MAP2K3 BC032478, NM 145109 -2.23 MAPKAPK5 NM003668,NM139078 -2.28 MARS NM004990 -2.41 MAZ AF489858, BC041629, L01420, M94046 -2.3 MCM2 D83987, NM 004526 -3.33 MCM3 NM_002388 -2.31 MCM5 NM_006739 -2.85 MCM7 AF279900, BC009398, BC013375, NM_005916, NM 182776 -2.95 MDH2 NM 005918 -2.99 MED6 NM 005466 -2.19 MED8 NM 001001651, NM 001001654, NM 052877, NM_201542 -2.36 MFAP4 NM 002404 4.43 MGAM NM 004668 2.32 MGAT4A N'M 012214 2.27 MGC14817 NM 032338 -4.52 MGC15416 NM 032371 -2.71 MGC2198 NM 138820 -2.79 MGC3121 NM 024031 -2.65 MGC34032 BC028743, NM 152697 4.33 MGC40157 NM 152350 -4.69 MGC52010 NM 194326 -3.16 MGC7036 NM 145058 2.49 MIBI AY147849, BC022403, NM 020774 -3.08 MIF NM 002415 -2.57 MIR16 BC012153, NM 016641 -2.51 MLC1 BC028425, NM 139202 4.78 MLF2 NM 005439 -2.62 MLX NM 170607, NM 198204 -2.05 MMP21 NM 147191 2.15 MRPL12 AF105278, NM 002949 -3.4 MRPL21 NM 181514 -3.19 MRPL23 NM 021134 -3.25 MRPL27 NM 016504 -3.71 MRPL35 NM 145644 2.94 MRPL37 AY421759, NM 016491 -2.92 MRPL51 NM 016497 2.85 MRPS2 NM 016034 -2.72 MRPS27 BC064902, NM_015084 4.25 MSH2 NM 000251 -2.16 MTCP1 CR600926, NM_014221 2.38 MTSS 1 AK027015, BC023998 2.42 MTVRI BC023991, CR610230, NM 152832 4.71 MUC17 AJ606307, NM_001004430 2.19 MUSTNI NM 205853 3.94 MYBL2 NM 002466 -3.25 MYOIO AB018342, AL832428, NM_012334 2.68 NALP12 AK095460, AY116204, AY116205, AYI 16207, NM 144687 2.49 NAPA AK126519, NM_003827 -2.11 NCOR2 AF113003, AK127788 -2.64 NDUFAIO NM 004544 -2.97 NDUFB 10 BC007509, NM_004548 -2.93 NDUFS8 NM 002496 -3.04 NDUFVI BC008146, CR624895, NM 007103 -3.4 NES NM 006617 4.6 NFATC3 NM 173164 -2.28 NFIX NM 002501 2.22 NID BC045606, NM_002508 2.9 NLGN4X AX773938, AY358562, NM 020742 2.05 NMEl NM 000269, NM 198175 -3.78 NOB1P BC064630, NM_014062 -2.68 NOLA2 NM 017838 -2.43 NP AK098544, AK126154, CR608316 -2.69 NPEPPS NM 006310, Y07701 -3.3 NRXN3 AJ316284, A7493127, AK056530, NM 138970 -2.93 NSEPI NM 004559 -4.05 NUP205 NM 015135 -2.49 NUP210 AB020713, NM_024923 -2.52 NUTF2 NM 005796 -3.57 OCIADI AF324350, NM 017830 -2.73 OCLN NM 002538 2.23 0R2L2 NM 001004686 2.56 P4HB BC029617, NM000918 -3.75 PA2G4 BC069786, NM 006191 -4.4 PABPC4 BC065540, BC071591, NM 003819 -2.77 PAF53 AK091294, NM 022490 -2.77 PARK7 NM 007262 -2.19 PCBP1 NM 006196 -3.81 PCBP2 AB188306, AB208825, NM_005016, NM 031989, X78136 -3.42 PCSKIN NM 013271 -2.23 PDXK BC000123, BC005825 -2.41 PECI AB209917, AF244138, BC002668, BC034702, NM_006117, 2 25 PFKFB2 BC069583 2.47 PFN1 NM 005022 -4.27 PGK1 NM 000291 -3.11 PGK2 NM 138733 2.18 PHEMX AB029488, AK128812, BC016693, NM_139022 4.56 PHF5A NM 032758 3.44 PKM2 NM 002654, NM_182470 -4.02 PLDN AK057545, AK091740 -3.06 PLTP NM006227, NM_182676 4.65 PNCK BC064422, CR611192, NM_198452 -3.12 POLD2 NM_006230 -2.73 POLE3 NM_017443 -5.14 POU3F2 NM_005604 2.27 PPHLNI AK124921, BC025306, NM 016488 3.83 PPM1G BC000057, NM_177983 -2.7 PPPICA CR595463, NM_001008709, NM 002708 -4.32 PPP1R10 NM 002714 2.05 PPPIR3D NM 006242 3.55 PPP2R2C BC032954, NM 020416, NM 181876 2.23 PQBPI AB041833, NM 005710 -2.05 PRDXI NM 002574 -2.87 PRDX5 AF124993, NM 012094 -3.08 PRKCSH NM 002743 -2.3 PRSS15 AK096626, AK127867, NM 004793, X74215, X76040 -2.42 PRSS16 AK126160, NM 005865 2.88 PRTN3 M29142, NM 002777 -2.55 PSENEN NM 172341 -2.28 PSIP1 BC064135, NM 021144 2.64 PSMA3 NM 002788, NM 152132 -3.79 PSMBI BC020807 -2.49 PSMB3 NM 002795 -3.5 PSMB4 NM 002796 -4.54 PSMB8 NM 004159, NM 148919 -3.33 PSMC3 NM 002804 -2.35 PSMC5 NM 002805 2.94 PSMF1 BC029836, CR592856, NM 006814 -2.69 PTCH2 AF119569, NM 003738 4.17 PTD008 NM 016145 -2.93 PTOV1 AY358168, BC042921, NM 017432 -2.33 PUSI AF318369, NM025215 3.01 PVRL4 AF218028, NM 030916 2.04 QARS AF130067, BC000394, NM_005051 -2.36 QTRTDI NM 024638 2.79 RAB40C AY823398, NM 021168 2.9 RABAC1 NM 006423 -2.26 RABGGTB NM 004582 -2.37 RAD51L1 BX248061, NM_133509 2.03 RALB AK127675, NM_002881 -2.16 RANBP17 AJ288953, AJ288954, AK027880, NM 022897 -2.08 RASGRP2 AK092882, NM005825, NM_153819 -2.8 RASGRP3 AB020653, NM 170672 2.06 RBBP4 NM 005610 -3.65 RBM3 AK026664, AY203954, NM 006743 -2.87 RBM6 AK124030, BC046643, NM 005777 -2.22 RBP3 J03912, NM_002900 -2.23 RFC2 NM 002914, NM 181471 -3.01 RFXANK CR622780, NM 003721, NM 134440 -2.97 RHBDLI AJ272344, NM_003961 4.81 RHOA NM 001664 -4.04 RHOG NM 001665 -2.7 RHOT2 AK090426, NM_138769 3.68 RKHD1 AB107353, NM 203304 -2.08 RNF144 NM 014746 3.24 RNF186 NM 019062 2.42 RNH NM 002939 6.22 RNPEP NM 020216 -3.57 RPILI AK127545, NM 178857 2.12 RPL11 BC018970, NM 000975 -2.2 RPL14 BC029036, NM 003973 -2.13 RPL18 NM 000979 -4.27 RPL18A NM 000980 -6.02 RPL22 NM 000983 -2.27 RPL29 NM 000992 -2.19 RPL3 AY320405, NM 000967 -2.97 RPL5 AB208980, BC001882, NM_000969 -3.82 RPL6 BC022444, NM 000970 -2.69 RPL8 NM 033301 -5.39 RPN2 AK096243, NM 002951 -3.92 RPS14 NM 005617 -3.06 RPS 19 NM 001022 -2.96 RPS3 BC034149, BC071669, NM 001005 -4.08 RPS5 NM 001009 -3.23 RPS9 NM 001013 -4.54 RSL1D1 NM 015659 -3.37 RUVBLI NM 003707 -2.63 S82297 S82297 -2.64 SAFB2 NM 014649 5.33 SCGBICI NM_145651 2.06 SCN8A NM 014191 -2.15 SCN9A NM 002977 2.76 SEC10L1 NM 006544 -3.38 SELO AY324823, NM 031454 -2.66 SEPT10 BC020502, NM_144710, NM_178584 2.61 SEPT6 AF403061 -3.51 SERF2 BC008214, NM 005770 -4.61 SETDBI BC009362, D31891, NM 012432 -2.08 SFRP2 NM003013 2.85 SH3BGR NM_007341 3.69 SH3YL1 BC008374, BC008375, NM 015677 2.28 SHMT2 BC011911, BC032584, NM 005412 -4.13 SIDT1 NM 017699 -2.84 SIMl NM_005068 2.28 SIVA AK128704, NM 006427, NM_021709 -2.66 SLC16A3 NM 004207 -2.43 SLC22A4 NM 003059 2.16 SLC25A3 NM_005888, NM 213611, NM 213612 -2.77 SLC25A6 NM 001636 -4.89 SLC35E1 AK027850, BC062562, NM_024881 -2.3 SLC39A3 NM 144564 -2.69 SLC40A1 NM 014585 2.41 SLC6A13 NM016615 2.16 SLC7A1 NM_003045 2.28 AF510501, AF510502, NM_033262, NM_058240, NM_182932, SLC8A3 ~ 182933, NM 182936,NM 183002 3.82 SMARCB1 AK024025, NM 001007468, NM 003073 -2.27 SND1 BC017180, NM 014390 -3.44 SNRP70 BC001315, CR592978, NM 001009820, NM 003089 2.83 SNRPA NM 004596 -4.38 SNRPB NM 198216 -4.7 SNX17 NM 014748 -2.81 SPHK1 BC030553, NM_021972, NM182965 2.15 SRM NM 003132 -4.07 SSR2 BC000341, BX649192, CR600571, NM 003145 -3.46 SSR4 NM 006280 -3.35 STAR NM 000349 4.48 STIMI NM_003156 -2.14 AF038897, AF305817, AF428146, BC073876, NM 001001433, STX16 ~ 001001434, NM 003763 3.49 SULF2 AY358461, BC020962, NM 018837, NM 198596 2.06 SUPT16H NM 007192 -3.16 SiN420H1 BC012933, NM 017635 3.27 SYNCRIP AF155568, BC032643, BC040844 -4 SYT9 BC046367, NM 175733 2.76 TBCID2 AF318370, AK124772, BC028918, BC071978, NM 018421 2.11 TCEA3 AY540752, NM 003196 2.21 TCF2 NM_000458 2.05 TCPI NM 030752 -2.98 TEGT NM 003217 -2.29 TFDP1 NM 007111 -2.66 TGM6 AF540970, NM 198994 2.24 TH1L AJ238379, AK023310, NM 198976 -3.38 THEM2 NM_018473 2.81 THOC4 NM 005782 -3.1 TIGD5 BC032632, NM 032862 4.05 TIMM17B BC091473, NM 005834 -2.65 TIMM50 CR617826, NM 001001563 -4.13 TIMP1 BC000866, NM_003254 -2.69 TKT BC002433, NM 001064 -3.34 TM4SF5 NM 003963 2.91 TMEM49 NM 030938 -2.88 TMPRSS 11 E AF064819, NM_014058 2.27 TNFAIP2 NM 006291 2.37 TOMM22 NM 020243 -4.55 TOMM70A NM 014820 -2.27 TOP1 NM_003286 2.36 TPM3 AK056889, AK056921, AK092712, BC072428, BX648485, -3.5 TPST2 NM 001008566 -2.27 TRIM28 BC052986, NM 005762 -3.66 TRIM6 CR749260, NM001003818, NM_058166 2.37 TRIP3 NM 004773 -2.48 TRPM4 AJ575813, AY297046, NM 017636 2.78 TSC BC015221, NM 017899 -2.77 TTC11 NM 016068 -3.32 TTC19 AK025958, AK056878, NM 0 17775 -2.46 TUBA6 NM 032704 -2.67 TUBB BC007605, NM 178014 -3.23 TUFM BC001633,NM 003321, S75463 -3.29 U16258 U16258 2.05 U5-116KD BC002360, NM 004247 -2.96 U78723 U78723 -2.57 UBADC1 NM 016172 -2.64 UBEI AK097343, NM 003334, X52897 -3.72 UBE2L3 NM 003347 -3.22 UBE2M NM 003969 -3.39 UBE2NL NM 001012989 -2.88 UBE2S NM 014501 -3.25 UBE4B AF043117, BC093696, NM 006048 3.16 UGP2 NM 001001521, NM 006759 -2.35 UNC5B AY126437, NM 170744 2.19 UNQ473 NM 198477 2.52 UNQ9391 NM 198464 2.19 UQCRCI CR618343, NM_003365 -2.35 UQCRC2 NM_003366 -3.57 URP2 NM_031471, NM_178443 3.55 UVRAG NM003369 2.65 UXT NM_004182, NM_153477 -2.31 VAMPB NM003761 -2.61 VAPB AF086629, AK127252, AK128422, NM004738 2.4 VDAC1 NM003374 -4.67 VDAC2 BC000165, L08666, NM003375 -3.64 VGLL4 NM 014667 -3.21 VIM AK093924, NM_003380 -2.71 VIP NM_003381, NM_194435 2.29 WDR58 AK075330, BC050674, NM_024339 -2.02 WDR60 BC014491, NM_018051 2.42 WDR61 NM 025234 -2.93 WIG1 AK122768, NM 022470 -2.21 XAB2 BC007208, NM 020196 5.29 XRCC6 AK055786, CR456492, NM 001469 -3.91 YWHAB NM 006761 -2.37 YWHAH BC003047, NM_003405 -3.07 ZBTB1 BC050719, NM 014950 2.05 ZDHHC8 AK131238, BC053544, NM 013373 2.37 ZKSCANI NM 003439 -2.32 ZNF167 NM 025169 2.2 ZNF207 BC002372, BC008023, CR616570, NM 003457 -3.31 ZNF323 BC008490, NM 030899, NM 145909 2.53 ZNF407 NM 017757 2.31 ZNF436 NM030634 2.88 ZYX NM003461 -2.87 Negative fold change values in Table 4 indicate a reduction in mRNA levels for a given gene compared to that observed for the negative controls.

[00256] Negative fold change values in Table 2, 3 and 4 indicate a reduction in mRNA levels for a given gene compared to that observed for the negative controls.
[00257] The results demonstrate that let-7 expression altered the expression levels, by at least two-fold, of 558 genes (217 down-regulated, 341 up-regulated) in cells, 1035 genes (531 down-regulated, 504 up-regulated) in HepG2 cells and genes (441 down-regulated, 280 up-regulated) in HL-60 cells.

WO 2008/073922 PCT/iTS2007/087037 EXAMPLE 3:

[00258] Gene targets for binding of hsa-let-7a, hsa-let-7b, and hsa-let-7g were predicted using the proprietary algorithm miRNATargetTM (Asuragen) and are shown in Table 5, the content of all database submission incorporated herein by reference in its entirety, as presented on the filing date of this application.

Table 5. Target genes of hsa-let-7a, hsa-let-7b, and hsa-let-7g.
Gene Symbol RetSe Gene Name 2'-PDE NM 177966 2'- hos hodiesterase ABCB9 NM 019624 ATP-binding cassette, sub-family B (MDR/TAP, ABCC10 NM 033450 ATP-binding cassette, sub-faTnily C, member 10 ABCC5 NM 005688 ATP-binding cassette, sub-family C, member 5 ACSL6 NM 001009185 acyl-CoA synthetase long-chain family member 6 ACTR2 NM 001005386 actin-related protein 2 isoform a ACVRIB NM 004302 activin A type IB receptor isoform a precursor ACVR2A NM 001616 activin A receptor, type TIA precursor ADAM15 NM 207191 a disintegrin and metallo roteinase domain 15 ADAMTS5 NM 007038 ADAM metallo e tidase with thrombospondin type I
ADAMTS8 NM 007037 ADAM metallopeptidase with thrombospondin type I
ADCY9 NM 001116 adenylate cyclase 9 ADIPOR2 NM 024551 adiponectin receptor 2 ADRB2 NM 000024 adrenergic, beta-2-, receptor, surface ADRB3 NM 000025 adrenergic, beta-3-, receptor AHCTFI NM 015446 transcription factor ELYS
AKAP6 NM 004274 A-kinase anchor protein 6 ANGPTL2 NM 012098 angiopoietin-like 2 precursor ANKFYI NM 016376 ankyrin repeat and FYVE domain containing I
ANKRD43 NM 175873 ankyrin repeat domain 43 ANKRD49 NM 017704 fetal globin inducing factor AP1S1 NM 057089 adaptor-related protein complex 1, sigma l APBB3 NM 006051 amyloid beta precursor rotein-bindin , family APPBP2 NM 006380 amyloid beta precursor protein-binding protein ARHGAP20 NM 020809 Rho GTPase activating protein 20 ARHGAP28 NM 001010000 Rho GTPase activating protein 28 isoform a ARHGEF15 NM 173728 Rho guanine exchange factor 15 ARID3A NM 005224 AT rich interactive domain 3A (BRIGHT- like) ARID3B NM 006465 AT rich interactive domain 3B (BRIGHT- like) ARL5A NM 012097 ADP-ribosylation factor-like 5A isoform I
ARPP-19 NM 006628 cyclic AMP phosphoprotein, 19 kD
ASAH3L NM 001010887 N-acylsphingosine amidoh drolase 3 like ATG16L1 NM 017974 APG 16 autophagy 16-like isoform 2 ATP2A2 NM 170665 ATPase, Ca++ transporting, cardiac muscle, slow ATP2B1 NM 001001323 lasma membrane calcium ATPase I isoform P
ATP2B3 NM 021949 plasma membrane calcium ATPase 3 isoform 3' ATP2B4 NM 001001396 plasma membrane calcium ATPase 4 isoform 4' ATXNI NM 000332 ataxin I
BACH1 NM 001186 BTB and CNC homology 1 isoform a BCAP29 NM 001008405 B-cell receptor-associated protein BAP29 isoform BCL2L1 NM 001191 BCL2-like 1 isoform 2 BCL7A NM 001024808 B-cell CLL/lymphoma 7A isoform b BIN3 NM 018688 bridging integrator 3 BNC2 NM 017637 basonuclin 2 BRD3 NM 007371 bromodomain containing protein 3 BTBD3 NM 014962 BTB/POZ domain containing protein 3 isoform a BTG2 NM 006763 B-cell translocation gene 2 BZW 1 NM 014670 basic leucine zipper and W2 domains I
BZW2 NM 014038 basic leucine zipper and W2 domains 2 C10orf6 NM 018121 hypothetical protein LOC55719 Cl lorfl1 NM 006133 neural stem cell-derived dendrite regulator Cl lorf5l NM 014042 h othetical protein LOC25906 Cl lorf57 NM 018195 h othetical protein LOC55216 C15orf29 NM 024713 h othetical protein LOC79768 C15orf41 NM 032499 hypothetical protein LOC84529 Clorf22 NM 025191 hypothetical protein LOC80267 C21 orf29 NM 144991 chromosome 21 open reading frame 29 C22orf8 NM 017911 hypothetical protein LOC55007 C3orf64 NM 173654 AER61 lycos ltransferase C3orf9 NM 152305 hypothetical protein LOC56983 C6orfl2O NM 001029863 hypothetical protein LOC387263 C6orf211 NM 024573 hypothetical protein LOC79624 C8orf36 NM 173685 hypothetical protein LOC286053 C9orf28 NM 033446 hypothetical protein LOC89853 isoform 1 C9orf7 NM 017586 hypothetical protein LOC1 1094 CALD1 NM 004342 Caldesmon 1 isoform 2 CAP 1 NM 006367 adenyl 1 cyclase-associated protein CASP3 NM 004346 caspase 3 pr ro rotein CBL NM 005188 Cas-Br-M murine ecotropic retroviral CBX2 NM 005189 chromobox homolog 2 isoform I
CCND1 NM 053056 cyclinDl CCND2 NM 001759 c clin D2 CCNJ NM 019084 cyclin J
CCR7 NM 001838 Chemokine (C-C motif) receptor 7 precursor CD164 NM 006016 CD164 anti en sialomucin CDC25A NM 001789 cell division cycle 25A isoform a CDC34 NM 004359 cell division cycle 34 CDV3 NM 017548 CDV3 homolog CDYL NM 004824 chromodomain protein, Y chromosome-like isoform CEECAMI NM 016174 cerebral endothelial cell adhesion molecule 1 CEP164 NM 014956 hypothetical protein LOC22897 CGNLI NM 032866 cingulin-like 1 CHD7 NM 017780 chromodomain helicase DNA binding protein 7 CHD9 NM 025134 chromodomain helicase DNA binding protein 9 CHES1 NM 005197 checkpoint suppressor I
CLASP2 NM 015097 CLIP-associating protein 2 CLDN12 NM 012129 claudin 12 COIL NM 004645 Coilin COL14A1 NM 021110 colla en, type XIV, alpha 1 COL15A1 NM 001855 al ha 1 type XV collagen precursor COL19AI NM 001858 al ha 1 type XIX collagen precursor COLIAI NM 000088 al ha 1 type I collagen re ro rotein COL1A2 NM 000089 alpha 2 type I collagen COL24A1 NM 152890 colla en, type XXIV, al ha 1 COL3A1 NM 000090 procoliMen, type III, alpha 1 COL4A1 NM 001845 alpha 1 type IV collagen re ro rotein COIt1A5 NM 000495 alpha 5 type IV collagen isoform 1, precursor COL5A2 NM 000393 al ha 2 type V colla en preproprotein CPA4 NM 016352 carbox e tidase A4 preproprotein CPD NM 001304 carboxypeptidase D precursor CPEB2 NM 182485 cytoplasmic polyadenylation element binding CPEB3 NM 014912 cytoplasmic polyadenylation element binding CPEB4 NM 030627 c o lasmic polyadenylation element binding CPM NM 001005502 carbox e tidase M precursor CPSF4 NM 006693 cleavage and polyadenylation specific factor 4, CROP NM 016424 cisplatin resistance-associated overexpressed CRTAP NM 006371 cartilage associated protein precursor CTDSPL2 NM 016396 CTD carboxy-terminal domain, RNA polyinerase II, CTNS NM 004937 Cystinosis, ne hro athic isoform 2 CTSC NM 148170 cathepsin C isoform b precursor CYP19A1 NM 000103 cytochrome P450, family 19 DCUNiD2 NM 001014283 h othetical protein LOC55208 isoform b DCUNID3 NM 173475 hypothetical protein LOC123879 DCX NM 000555 doublecortin isoform a DDI2 NM 032341 DNA-damage inducible protein 2 DDX19A NM 018332 DDX19-like protein DDX19B NM 001014449 DEAD (As -Glu-Ala-As) box polypeptide 19 isoform DDX19-DDX19L NM 001015047 DDX19-DDX19L protein DHX57 NM 198963 DEAH (As -Glu-Ala-As /His) box polypeptide 57 DKFZ 686K16132 NM 001012987 hypothetical protein LOC388957 DLC 1 NM 006094 deleted in liver cancer 1 isoform 2 DLST NM 001933 dihydrolipoamide S-succinyltransferase (E2 DMD NM 000109 D stro hin Dp427c isoform DMP1 NM 004407 dentin matrix acidic hos ho rotein DNAJCI NM 022365 DnaJ (Hs 40) homolog, subfamily C, member I
DOCK3 NM 004947 dedicator of cytokinesis 3 DPP3 NM 005700 di e tidyl eptidase III
DSCAM NM 206887 Down syndrome cell adhesion molecule isoform DST NM 015548 dystoninisoform IeA precursor DTX2 NM 020892 deltex 2 DUSPI NM 004417 dual specificity phosphatase 1 DUSP16 NM 030640 dual s ecificit phosphatase 16 DUSP9 NM 001395 dual s ecificit phosphatase 9 DYRKIA NM 001396 dual-s ecificity t osine- Y)- hosphorylation DZIP1 NM 014934 DAZ interacting protein 1 isoform 1 E2F5 NM 001951 E2F transcription factor 5 EFHD2 NM 024329 EF hand domain family, member D2 EIF2C4 NM 017629 Euka otic translation initiation factor 2C, 4 EIF4G2 NM 001418 Eukaryotic translation initiation factor 4 ELOVL4 NM 022726 Elongation of ve lon chain fatty acids EPHA3 NM 005233 ephrin receptor EphA3 isoform a precursor EPHA4 NM 004438 ephrin receptor EphA4 ERCC6 NM 000124 excision repair cross-complementing rodent ERGIC l NM 001031711 endoplasmic reticulum- ol i intermediate FAM104A NM 032837 hypothetical protein LOC84923 FAM84B NM 174911 breast cancer membrane protein 101 FAM96A NM 001014812 hypothetical protein FLJ22875 isoform b FARPI NM 005766 FERM, RhoGEF, and pleckstrin domain protein 1 FASLG NM 000639 fas ligand FBXL19 NM 019085 F-box and leucine-rich repeat protein 19 FGF11 NM 004112 fibroblast growth factor 11 FIGN NM 018086 Fidgetin FLJ20232 NM 019008 hypothetical protein LOC54471 FLJ20309 NM 017759 hypothetical protein LOC54891 FLJ21986 NM 024913 hypothetical protein LOC79974 FLJ25476 NM 152493 hypothetical protein LOC 149076 FLJ31818 NM 152556 hypothetical protein LOC154743 FLJ36031 NM 175884 hypothetical protein LOC168455 FLJ36090 NM 153223 hypothetical protein LOC153241 FLJ39779 NM 207442 hypothetical protein LOC400223 FLJ90709 NM 173514 hypothetical protein LOC153129 FNDC3A NM 014923 Fibronectin type III domain containin 3A
FNDC3B NM 022763 Fibronectin type III domain containing 3B
FRAS I NM 025074 Fraser syndrome 1 isoform 1 GAB2 NM 012296 GRB2-associated binding protein 2 isoform b GABPA NM 002040 GA binding protein transcription factor, alpha GALE NM 000403 UDP-galactose-4-epimerase GALNTI NM 020474 pol e tide N-acetyl alactosamin ltransferase I
GALNTL2 NM 054110 UDP-N-acetyl-al ha-D-galactosamine: ol eptide GAN NM 022041 Gigaxonin GAS7 NM 003644 growth arrest-specific 7 isoform a GCNT4 NM 016591 core 2 beta-1,6-N-acetyllucosaminyltransferase GDPDI NM 182569 glycero hos hodiester phosphodiesterase domain GGA3 NM 014001 ADP-ribosylation factor binding protein 3 GHR NM 000163 growth hormone receptor precursor GIPCI NM 005716 re lator of G- rotein signaling 19 interactin GM632 NM 020713 hypothetical protein LOC57473 GNAL NM 002071 guanine nucleotide binding protein (G protein), GNG5 NM 005274 guanine nucleotide binding protein (G protein), GNS NM 002076 glucosamine (N-acetyl)-6-sulfatase precursor GOLTIB NM 016072 golgi transport 1 homolog B
GPATC3 NM 022078 G patch domain containing 3 GPR137 NM 020155 hypothetical protein LOC56834 GTF21 NM 001518 general transcription factor II, i isoforni 4 HAND1 NM 004821 basic helix-loo -helix transcription factor HDHDIA NM 012080 haloacid dehalogenase-like hydrolase domain HDLBP NM 005336 high density li o rotein binding protein HEAB NM 006831 ATP/GTP-bindin protein HECTD2 NM 182765 HECT domain containing 2 isoform a HIC2 NM 015094 hypermethylated in cancer 2 HK2 NM 000189 hexokinase 2 HMGA2 NM 001015886 high mobility group AT-hook 2 isoform c HOMER2 NM 199331 homer 2 isoform 3 HOXAI NM 153620 Homeobox Al isoform b HOXA9 NM 152739 Homeobox A9 HOXCI 1 NM 014212 Homeobox C11 HOXD1 NM 024501 Homeobox D1 HTR4 NM 000870 5-hydroxytryptamine (serotonin) receptor 4 IDH2 NM 002168 isocitrate dehydrogenase 2 (NADP+), IGF2BPI NM 006546 insulin-like growth factor 2 mRNA binding IGF2BP2 NM 001007225 insulin-like growth factor 2 mRNA binding IGF2BP3 NM 006547 insulin-like owth factor 2 mRNA binding IKBKAP NM 003640 inhibitor of kappa light polypeptide gene IKBKE NM 014002 IKK-related kinase epsilon ILIO NM 000572 Interleukin 10 precursor IL6 NM 000600 Interleukin 6 (interferon, beta 2) INPP5A NM 005539 inositol polyphosphate-5-phosphatase A
IRS2 NM 003749 insulin receptor substrate 2 ITGB3 NM 000212 integrin beta chain, beta 3 precursor ITSNI NM 001001132 Intersectin I isoform ITSN-s JMJDIA NM 018433 jumon'idomaincontaininlA
KIAA0179 NM 015056 hypothetical protein LOC23076 KIAA0664 NM 015229 hypothetical protein LOC23277 KIAA1539 NM 025182 h othetical protein LOC80256 KIAA1961 NM 001008738 hypothetical protein LOC96459 isoform 2 KIF2 NM 004520 kinesin heavy chain member 2 KLF9 NM 001206 Krup el-like factor 9 KLHL6 NM 130446 kelch-like 6 KPNA4 NM 002268 karyopherin alpha 4 LBH NM 030915 hypothetical protein DKFZ 566J091 LEPROTLI NM 015344 leptin receptor overla in transcript-like 1 LGR4 NM 018490 leucine-rich repeat-containing G protein-coupled LIMD 1 NM 014240 LIM domains containing 1 LIMD2 NM 030576 hypothetical protein LOC80774 LIN28B NM 001004317 lin-28 homolog B
LNK NM 005475 lym hoc e adaptor protein LOC144097 NM 138471 hypothetical rotein LOC144097 LOC220594 NM 145809 TL132 protein LOC51136 NM 016125 PTD016 protein LOXL4 NM 032211 lysyl oxidase-like 4 precursor LPGAT1 NM 014873 lyso hos hatid 1 lycerol acyltransferase 1 LRIG2 NM 014813 leucine-rich repeats and immunoglobulin-like LRIG3 NM 153377 leucine-rich repeats and immunoglobulin-like LRRCI NM 018214 leucine rich repeat containing 1 LRRC17 NM 005824 leucine rich repeat containing 17 isoform 2 LRRFIP 1 NM 004735 leucine rich repeat (in FLII interacting LSM11 NM 173491 LSM11, U7 small nuclear RNA associated LYPLA3 NM 012320 lyso hos holi ase 3 (lysosomal phospholipase MAP3K3 NM 002401 mitogen-activated protein kinase kinase kinase 3 MAP3K7IP2 NM 015093 mitogen-activated protein kinase kinase kinase 7 MAP4K3 NM 003618 mitogen-activated protein kinase kinase kinase MAPK6 NM 002748 mitogen-activated protein kinase 6 MARCH9 NM 138396 Membrane-associated RING-CH protein IX
MDFI NM 005586 MyoD family inhibitor MECP2 NM 004992 methyl CpG binding protein 2 MED6 NM 005466 mediator of RNA polymerase II transcription, MEF2D NM 005920 MADS box transcription enhancer factor 2, MEIS2 NM 002399 Homeobox protein Meis2 isoform f MEIS3 NM 001009813 Meisl, myeloid ecotropic viral integration site MGAT4A NM 012214 Mannosyl (al ha-1,3-) I co rotein MGC17330 NM 052880 HGFL protein MGC61598 NM 001004354 hypothetical protein LOC441478 MGLL NM 001003794 monoglyceride lipase isoform 2 MIB 1 NM 020774 Mindbomb homolog 1 MLL5 NM 182931 myeloid/lymphoid or mixed-lineage leukemia 5 MLLT10 NM 001009569 myeloid/lymphoid or mixed-lineage leukemia MLRI NM 153686 transcription factor MLRl MLR2 NM 032440 li and-dependent corepressor MMP11 NM 005940 matrix metalloproteinase 11 preproprotein MNT NM 020310 MAX binding protein MTPN NM 145808 M otro hin MYCLI NM 001033081 1-m c-1 proto-oncogene isoform 1 MYCN NM 005378 v-myc myelocytomatosis viral related oncogene, MYRIP NM 015460 myosin VIIA and Rab interacting protein NAB 1 NM 005966 NGFI-A binding protein 1 NAPILl NM 004537 nucleosome assembly protein 1-like 1 NAT12 NM 001011713 hypothetical protein LOC122830 NAT5 NM 181528 N-acetyltransferase 5 isoform c NCOAI NM 003743 nuclear receptor coactivator 1 isoform 1 NCOA3 NM 006534 nuclear receptor coactivator 3 isoform b NDST2 NM 003635 N-deacetylase/N-sulfotransferase (heparan NID2 NM 007361 nidogen 2 NKIRAS2 NM 001001349 NFKB inhibitor interacting Ras-like 2 NME4 NM 005009 Nucleoside-di hos hate kinase 4 NME6 NM 005793 Nucleoside di hos hate kinase type 6 NOVAl NM 002515 neuro-oncological ventral antigen 1 isoform 1 NRAS NM 002524 neuroblastoma RAS viral (v-ras) oncogene NRK NM 198465 Nik related kinase NUMBL NM 004756 numb homolog (Droso hila -like NUP98 NM 005387 nucleoporin 98kD isoform 3 NXT2 NM 018698 nuclear transport factor 2-like export factor 2 OLR1 NM 002543 oxidised low density li o rotein (lectin-like) OSBPL3 NM 015550 oxysterol-binding protein-like protein 3 isoform OSMR NM 003999 Oncostatin M receptor P18SRP NM 173829 P18SRP protein P4HA2 NM 001017973 rol 14-hydroxylase, alpha II subunit isoform 2 PAK1 NM 002576 21-activated kinase 1 PANX2 NM 052839 pannexin 2 PAPPA NM 002581 Pre anc -associated plasma protein A
PAX3 NM 181457 paired box gene 3 isoform PAX3 PBX2 NM 002586 pre-B-cell leukemia transcription factor 2 PBX3 NM 006195 re-B-cell leukemia transcription factor 3 PCDH19 NM 020766 protocadherin 19 PCGF3 NM 006315 ring finger protein 3 PCYTIB NM 004845 Phosphate cytidylyltransferase 1, choline, beta PGM2L1 NM 173582 phos ho ucomutase 2-like I
PGRMCI NM 006667 progesterone receptor membrane component 1 PHF8 NM 015107 PHD finger protein 8 PIGA NM 002641 phosphatidylinositol PLCXD3 NM 001005473 hos hatidylinositol-s ecific phospholipase C, X
PLDN NM 012388 Pallidin PLEKHG6 NM 018173 pleckstrin homology domain containing, family G

PLXNDI NM 015103 plexin D1 POM121 NM 172020 nuclear pore membrane protein 121 PPAPDC2 NM 203453 phosphatidic acid phosphatase type 2 domain PPARGCIA NM 013261 peroxisome proliferative activated receptor PPP1R12B NM 002481 protein phosphatase 1, regulatory (inhibitor) PPP1R15B NM 032833 protein phosphatase 1, regulatory subunit 15B
PPP1R16B NM 015568 protein phosphatase I re ulato inhibitor PPP3CA NM 000944 protein phosphatase 3(formerl 2B), catalytic PRDM2 NM 001007257 retinoblastoma protein-binding zinc finger PREI3 NM 015387 preimplantation protein 3 isoform 1 PRPF38B NM 018061 PRP38 re-mRNA processing factor 38 (yeast) PSCD3 NM 004227 Pleckstrin homology, Sec7 and coiled/coil PSD3 NM 015310 ADP-ribosylation factor guanine nucleotide PYGO2 NM 138300 pygopus homolog 2 PYY2 NM 021093 peptide YY, 2 (seminalplasmin RAB11FIP4 NM 032932 RAB11 family interacting protein 4 (class II) RAB 15 NM 198686 Ras-related protein Rab-15 RAB40C NM 021168 RAR (RAS like GTPASE) like RAI16 NM 022749 retinoic acid induced 16 RALB NM 002881 v-ral simian leukemia viral oncogene homolog B
RALGPS 1 NM 014636 Ral GEF with PH domain and SH3 binding motif I
RANBP2 NM 006267 RAN binding protein 2 RASLIOB NM 033315 RAS-like, family 10, member B
RAVER2 NM 018211 ribonucleoprotein, PTB-binding 2 RB 1 NM 000321 retinoblastoma 1 RBM9 NM 001031695 RNA binding motif protein 9 isoform 1 RDH10 NM 172037 retinol deh dro enase 10 REEP1 NM 022912 receptor expression enhancing protein 1 RFXDC 1 NM 173560 Regulatory factor X domain containing 1 RGAGl NM 020769 retrotransposon gag domain containing I
RGS16 NM 002928 regulator of G-protein signalling 16 RICTOR NM 152756 Rapamycin-insensitive companion of mTOR
RIOK3 NM 003831 sudD suppressor of bimD6 homolog isoform 1 RNF38 NM 022781 ring finger protein 38 isoform I
RNF44 NM 014901 ring finger protein 44 RNF5 NM 006913 ring finger protein 5 RNF7 NM 014245 ring finger protein 7 isoform 1 RNPC1 NM 017495 RNA-binding region containing protein 1 isoform RORC NM 001001523 RAR-related orphan receptor C isoform b RPS6KA3 NM 004586 ribosomal protein S6 kinase,90kDa, polypeptide RRM2 NM 001034 ribonucleotide reductase M2 ol e tide RRP22 NM 001007279 RAS-related on chromosome 22 isoform b RSPO2 NM 178565 R-spondin family, member 2 RUFY3 NM 014961 ra 2 interactin protein x isoform 2 SBKI NM 001024401 SH3-binding domain kinase 1 SCN5A NM 000335 voltage-gated sodium channel type V al ha SCUBE3 NM 152753 signal peptide, CUB domain, EGF-like 3 SEC14L1 NM 003003 SEC14 (S. cerevisiae)-like 1 isoform a SEC24C NM 004922 SEC24-related protein C
SEMA3F NM 004186 semaphorin 3F
SENP2 NM 021627 SUMO1/sentrin/SMT3 specific protease 2 SENP5 NM 152699 SUMO1/sentrin specific protease 5 SFRS12 NM 139168 s licin factor, arginine/serine-rich 12 SFRS8 NM 152235 splicing factor, arginine/serine-rich 8 isoform SGCD NM 000337 delta-sarcoglycan isoform 1 SLC20A1 NM 005415 solute carrier family 20 (phosphate SLC25A18 NM 031481 solute carrier SLC25A24 NM 013386 solute carrier family 25 member 24 isoform I
SLC25A27 NM 004277 solute carrier family 25, member 27 SLC25A4 NM 001151 solute carrier family 25 (mitochondrial carrier;
SLC26A9 NM 052934 solute carrier family 26, member 9 isoform a SLC30A4 NM 013309 solute carrier family 30 (zinc trans orter , SLC5A6 NM 021095 solute carrier family 5 (sodium-deendent SLC6A1 NM 003042 solute carrier family 6(neurotransmitter SLC9A9 NM 173653 solute carrier family 9 (sodium/hydrogen SLCO5A1 NM 030958 organic anion transporter ol e tide-related SMARCADI NM 020159 SWI/SNF-related, matrix-associated SNAP23 NM 003825 synaptosomal-associated protein 23 isoform SNN NM 003498 Stannin SNX16 NM 022133 sorting nexin 16 isoform a SOCS1 NM 003745 Suppressor of cytokine signaling 1 SOCS4 NM 080867 Suppressor of cytokine signaling 4 SOX13 NM 005686 SRY-box 13 SPATA2 NM 006038 spermatogenesis associated 2 SPRYD4 NM 207344 hypothetical protein LOC283377 STARD3NL NM 032016 MLN64 N-terminal homolog STAT3 NM 213662 signal transducer and activator of transcription STK40 NM 032017 SINK-homologous serine/threonine kinase STRBP NM 018387 Spermatid perinuclear RNA-binding protein STX17 NM 017919 syntaxin 17 STX3A NM 004177 syntaxin 3A
STXBP5 NM 139244 Tomosyn SURF4 NM 033161 surfeit 4 SYT1 NM 005639 synaptotagmin I
SYTI1 NM 152280 syna tota min 12 TARBP2 NM 134324 TAR RNA binding protein 2 isoform b TBKBPI NM 014726 ProSAPiP2 protein TBX5 NM 000192 T-box 5 isoform 1 TMED5 NM 016040 transmembrane emp24 protein transport domain TMEM65 NM 194291 hypothetical protein LOC157378 TMPRSS2 NM 005656 transmembrane protease, serine 2 TNFRSFIB NM 001066 tumor necrosis factor receptor 2 precursor TOB2 NM 016272 Transducer of ERBB2, 2 TPP1 NM 000391 Tripeptidyl- e tidase I precursor TRHDE NM 013381 thyrotropin-releasing hormone de radin enzyme TRIB 1 NM 025195 G-protein-coupled receptor induced protein TRIB2 NM 021643 tribbles homolog 2 TRIM33 NM 015906 tripartite motif-containing 33 protein isoform TRIM41 NM 033549 tripartite motif-containing 41 isform 1 TRPM6 NM 017662 transient receptor potential cation channel, TSC22D2 NM 014779 TSC22 domain family 2 1TL NM 153712 tubulin tyrosine ligase TTLLEI NM 014640 tubulin tyrosine ligase-like family, member 4 TUSC2 NM 007275 tumor suppressor candidate 2 UBXD2 NM 014607 UBX domain containing 2 UGCGLI NM 001025777 UDP-glucose ceramide glucosyltransferase-like 1 UHRF2 NM 152896 Np95-like ring finger protein isoform b ULK2 NM 014683 unc-51-like kinase 2 UNC5A NM 133369 netrin receptor Unc5h1 USP21 NM 001014443 ubi uitin-s ecific protease 21 USP32 NM 032582 ubiguitin specific protease 32 USP47 NM 017944 ubiquitin specific protease 47 VANGL2 NM 020335 vang-like 2 (van gogh, Droso hila VCPIPI NM 025054 valosin containin protein (p97)/p47 complex VSNL1 NM 003385 visinin-like 1 WDFY3 NM 014991 WD repeat and FYVE domain containing 3 isoform WNTI NM 005430 win less-t e MMTV integration site family, XKR8 NM 018053 X Kell blood group precursor-related family, YOD l NM 018566 h othetical protein LOC55432 ZBTB10 NM 023929 zinc fmger and BTB domain containing 10 ZBTB39 NM 014830 zinc fmger and BTB domain containing 39 ZBTB5 NM 014872 zinc finger and BTB domain containing 5 ZCCHC5 NM 152694 zinc finger, CCHC domain containing 5 ZFYVE26 NM 015346 zinc fmger, FYVE domain containing 26 ZMATI NM 001011656 zinc finger, matrin type 1 isoform 2 ZNF294 NM 015565 zinc fmger protein 294 ZNF644 NM 016620 zinc f er protein 644 isoform 2 ZNF710 NM 198526 zinc fin er protein 710 ZNF740 NM 001004304 zinc finger protein 740 ZSWIM4 NM 023072 zinc finger, SWIM domain containing 4 [00259] The predicted gene targets that exhibited altered mRNA expression levels in HepG2 and A549 cells, following transfection with pre-miR hsa-let-7b, are shown in Table 6 below.

Table 6. Predicted hsa-let-7 targets that exhibited altered mRNA expression levels in HepG2 and A549 cells 72 hrs after transfection with pre-miR hsa-let-7b.
Gene Symbol RefSe Gene Name Expression Altered in HepG2 & A549 2'-PDE NM 177966 2'-phosphodiesterase ACVRIB NM 004302 activin A type IB receptor isoform a precursor C6orf2I1 NM 024573 hypothetical protein LOC79624 CDC25A NM 001789 cell division cycle 25A isoform a CDC34 NM_004359 cell division cycle 34 CHD7 NM_017780 chromodomain helicase DNA binding protein 7 COL4A5 NM_000495 alpha 5 type IV collagen isoform 1, precursor E2F5 NM001951 E2F transcription factor 5 FIGN NM_018086 Fidgetin GALE NM_000403 UDP-galactose-4-epimerase GNG5 NM 005274 guanine nucleotide binding protein (G protein), HDHDIA NM 012080 haloacid dehalogenase-like hydrolase domain HMGA2 NM_001015886 high mobility group AT-hook 2 isoform c KIAA0179 NM_015056 hypothetical protein LOC23076 LEPROTLI NM015344 leptin receptor overlapping transcript-like I
LIN28B NM_001004317 Lin-28 homolog B
MED6 NM_005466 mediator of RNA polymerase II transcription, NAPILI NM004537 nucleosome assembly protein 1-like I
NME6 NM_005793 nucleoside diphosphate kinase type 6 NRAS NM_002524 neuroblastoma RAS viral (v-ras) oncogene NUP98 NM_005387 nucleoporin 98kD isoform 3 PGRMC 1 NM_006667 progesterone receptor membrane component 1 PIGA NM_002641 phosphatidylinositol SLC25A24 NM_013386 solute carrier family 25 member 24 isoform. l SLC5A6 NM_021095 solute carrier family 5 (sodium-dependent SNAP23 NM 003825 synaptosomal-associated protein 23 isoform Expression Altered in HepG2 Only ARID3A NM_005224 AT rich interactive domain 3A (BRIGHT- like) ARL5A NM_012097 ADP-ribosylation factor-like 5A isoform I
C10orf6 NM_018121 hypothetical protein LOC55719 CCNJ NM 019084 cyclin 7 COIL NM 004645 coilin CPEB2 NM182485 cytoplasmic polyadenylation element binding CTDSPL2 NM_016396 CTD (carboxy-terminal domain, RNA polymerase H, CTSC NM_148I70 cathepsin C isoform b precursor DDX 19A NM_018332 DDX 19-like protein DLC 1 NM_006094 deleted in liver cancer 1 isoform 2 DMD NM_000109 dystrophin Dp427c isoform DST NM_015548 dystonin isofonn IeA precursor DUSP9 NM_001395 dual specificity phosphatase 9 DZIPI NM_014934 DAZ interacting protein I isoform I
EIF2C4 NM_017629 eukaryotic translation initiation factor 2C, 4 FLJ21986 NM_024913 hypothetical protein LOC79974 GNS NM_002076 glucosamine (N-acetyl)-6-sulfatase precursor HEAB NM 006831 ATP/GTP-binding protein HIC2 NM_015094 hypermethylated in cancer 2 IGF2BP1 NM_006546 insulin-like growth factor 2 mRNA binding LRIG3 NM_153377 leucine-rich repeats and immunoglobulin-like LSM11 NM_173491 LSM11, U7 small nuclear RNA associated MAPK6 NM_002748 mitogen-activated protein kinase 6 MGAT4A NM_012214 mannosyl (alpha-1,3-)-glycoprotein NAB 1 NM 005966 NGFI-A binding protein 1 PCYTIB NM 004845 phosphate cytidylyltransferase 1, choline, beta RAB 11FIP4 NM_032932 RAB 11 family interacting protein 4 (class II) RPS6KA3 NM 004586 ribosomal protein S6 kinase, 90kDa, polypeptide RRM2 NM001034 ribonucleotide reductase M2 polypeptide SLC20A1 NM_005415 solute carrier family 20 (phosphate SOCS1 NM003745 suppressor of cytokine signaling I
STK40 NM_0320I7 SINK-homologous serine/threonine kinase STX3A NM_004177 syntaxin 3A
TRIB2 NM_021643 tribbles homolog 2 TTLL4 NM_014640 tubulin tyrosine ligase-like family, member 4 TUSC2 NM_007275 tumor suppressor candidate 2 YODI NM_018566 hypothetical protein LOC55432 Expression Altered in A549 Only AP1S1 NM 057089 adaptor-related protein complex 1, sigma I
ATP2B4 NM_001001396 plasma membrane calcium ATPase 4 isoform 4a C6orf120 NM_001029863 hypothetical protein LOC387263 CD164 NM006016 CD164 antigen, sialomucin DUSP16 NM_030640 dual specificity phosphatase 16 FAM96A NM001014812 hypothetical protein FLJ22875 isoform b FLJ36031 NM175884 hypothetical protein LOC168455 FLJ90709 NM173514 hypothetical protein LOC153129 GOLTIB NM_016072 golgi transport 1 homolog B
GTF21 NM_001518 general transcription factor II, i isoform 4 HOXAI NM_153620 homeobox Al isoform b LGR4 NM_018490 leucine-rich repeat-containing G protein-coupled LPGATI NM_014873 lysophosphatidylglycerol acyltransferase 1 MTPN NM_145808 myotrophin NME4 NM_005009 nucleoside-diphosphate kinase 4 P18SRP NM_173829 P18SRP protein PGM2L1 NM_173582 phosphoglucomutase 2-like 1 STARD3NL NM_032016 MLN64 N-terminal homolog TMED5 NM_016040 transmembrane emp24 protein transport domain ZNF294 NM_015565 zinc finger protein 294 [00260] The data indicate that these predicted targets of hsa-let-7 exhibit altered mRNA expression within 72 hours of hsa-let-7b transfection into A549 or HepG2 cells. Under these experimental conditions, 26 predicted gene targets had altered mRNA levels in both cell types, 37 additional predicted gene targets had altered mRNA levels in HepG2 cells only, and twenty additional gene targets had altered mRNA levels in A549 cells only.

EXAMPLE 4:

[00261) Over-expression of hsa-let-7 in A549 and HepG2 cells results in the mis-regulation of numerous genes associated with cell division, cell proliferation, and the cell cycle. A list of those genes, their gene products, and associated protein functions are shown in Table 7.

Table 7. Cell cycle, cell division, cell proliferation, and DNA
synthesis/replication genes, gene products, and gene functions that respond to excess hsa-let-7.
Gene Product Function Gene expression reduced in HepG2 & A549 CCNA2 Binds CDK2 and CDC2 to promote cell cycle G 1/S and G2/M
cyclin A2 phase transition; aberrantly expressed in acute myeloid and romyelocytic leukemias CDC25A Cell division binds cyclins and regulates G1-S phase transition, over cycle 25A, a expressed in many cancers protein tyrosine-threonine phosphatase CDC34 cell division modifies CDKNIB increases the ubiquitination and defective 34 degradation of CDKN 1 B
ASK/DBF4 activator of S- Binds to and activates kinase activity of CDC7, required for the phase kinase initiation of DNA replication at the GI to S transition AURKA/STK6 Aurora A and maximally expressed during G2/M phases and may function in AURKB/STK12 Aurora B kinases c okinesis, up regulation in multiple neoplasms E2F5 E2F oncogenic in primary rodent cells and is amplified in human transcription breast tumors factor 5 CDK8 cyclin-dependent forms a complex with cyclin C that phosphorylates cyclin H
kinase 8 (CCNH), plays a role in the regulation of transcription and as component of the RNA olymerase II holoenzyme PLAGLI & pleomorphic transcription activators, regulate cell proliferation PLAGL2 adenoma gene-like transcription factors LIN28 homologue of Putative RNA binding protein heterochronic DICERI RNaseIII RNase processes pre-miRNAs and dsRNA
GMNN Geminin Geminin, regulates DNA replication and proliferation, binds to the licensing factor CDT1 and negatively regulates its ubiquitination, up regulated in breast, colon, rectal, and biliary tract neoplasms CHEKl checkpoint required for mitotic G2 checkpoint in response to radiation-homolog 1 induced DNA damage, associated with lung cancer Kinase NRAS Ras GTPase signaling molecule, mutated in multiple tumors Gene expression reduced in HepG2 only CDC2 call division binds B-type cyclins, regulates G2 to M phase transition, cycle 2, a cyclin romotes cell proliferation de endentkinase CCNB 1 cyclin B 1 regulatory subunit of the CCNB 1- CDC2 maturation-promoting factor complex that mediates G2-M phase transition, up-regulated in various cancers CCNE2 cyclin E2 GI-specific cyclin-dependent kinase regulatory subunit that interacts with CDK2 and CDK3, over-expressed in transformed cells and up regulated in breast and lung cancer CCNF cyclin F a member of the cyclin family of CDK kinase regulatory subunits, forms a complex with cyclin B1 (CCNB1) and CDC2 CCNJ cyclin J Protein containing cyclin C-terminal and N-terminal domains, has a region of low similarity to a region of cyclin A2 (human CCNA2) SKP2 S-phase kinase - a component of a ubiquitin E3 ligase complex, mediates cell associated cycle regulatory protein degradation, promotes cell protein 2 proliferation and invasion, inhibits cell adhesion and apoptosis;
over-expressed in many cancers CKSIB CDC28 protein Binds SKP2 and targets it to its substrates, required for kinase regulatory ubiquitination of p21 Cipl (CDKNIA) and p27 Kipl subunit 1B (CDKNIB), highly expressed in non-small cell lung, gastric, and colon carcinoma CDC20 cell division activates the mitotically phosphorylated form of the anaphase cycle 20 promoting complex as well as the mitotic spindle checkpoint, over-expressed in gastric cancer CDCAl cell division mediates stable attachment of microtubules to the kinetochore cycle associated during mitosis and play a role in the spindle checkpoint I
CDAC2 cell division Novel protein cycle associated CDAC3/ cell division a cytosolic protein that is degraded during G 1 phase and whose TOMEl cycle associated gene promoter activity is stimulated at the G2/M phase 3/ trigger of mitotic en 1 CDCA5 cell division Novel protein cycle associated CDAC7 cell division a nuclear protein expressed highly in thymus and small cycle associated intestine, has a role in anchorage-dependent growth, up 7 regulated in Burkitt lymphoma cell lines; corresponding gene may be a MYC target CDCA8 cell division a chromosomal passenger complex component, may target cycle associated survivin (BIRC5) and INCENP to centromere, required for 8 (borealin) kinetochore function, mitotic spindle stability, and metaphase chromosome alignment during mitosis RRM1 & ribonucleotide DNA synthesis RRM2 reductase M1 and M2 polypeptides CDC6 encoding cell DNA replication, up regulated in cervical intraepithelial division cycle 6 neoplasia and cervical cancer homologue CDC45L cell division associates with ORC2L, MCM7, and POLA2, predicted to be c cle 451ike involved in the initiation of DNA replication CDTI chromatin ensures replication occurs once per cell cycle, up regulated in licensing factor non small cell lung carcinomas ORC1L & origin DNA replication ORC6L recognition complex proteins MCM2/3/4/5 mini DNA replication, up-regulated in multiple cancers MCM6/7/8/10 chromosome maintenance deficient complex RFC2/3/4/5 replication factor DNA replication C complex E2F6 & E2F Regulators of cell cycle E2F8 transcription factors BUB 1& Budding acts in spindle assembly checkpoint and chromosome BUB1B uninhibited by congression, may regulate vesicular traffic; mutations are benzimidazoles associated with lung cancer, T cell leukemia and colorectal I homologs cancer cell chromosomal instability ; a protein kinase of the mitotic spindle checkpoint, inhibits anaphase-promoting complex activation, marker for colorectal cancer; mutation causes mosaic variegated aneuploidy with tumors MAD2L1 MAD2 mitotic component with BUB1B
arrest deficient-like 1 CDC23 cell division a putative component of the anaphase promoting complex cycle 23 (APC) which promotes the metaphase to anaphase transition, considered a tumor antigen in ovarian carcinoma; mutation in corresponding gene is associated with colon cancer FANCD2 Fanconi anemia involved in DNA damage response complementation roup D2 BRCAI & Breast Cancer tumor suppressors; mutations are linked to breast and ovarian BRCA2 Susceptibility cancer loci Gene expression increased in HepG2 & A549 CCNG2 cyclin G2 Down-regulated in thyroid papillary carcinoma RRM2B ribonucleotide DNA Synthesis, up regulated by p53 reductase M2B
Gene expression increased in HepG2 only CDKN2B cyclin-dependent interacts with the D type cyclin dependent kinases CDK4 and kinase inhibitor CDK6, inhibits cell proliferation; gene deletion and promoter 2B hypermeth lation are associated with many different neoplasms MXI1 MAX-interacting transcription regulator, antagonizes MYC, tumor suppressor in rotein 1 prostatic neoplasms [00262] These data indicate that hsa-let-7 is a key regulator of cell cycle progression. Many of the hsa-let-7-responsive genes are known oncogenes or are over-expressed in tumors. It is likely that in cancer cells with hsa-let-7 deletions or with reduced hsa-let-7 expression, many of these genes would be up-regulated, which would likely stimulate cell cycle and DNA synthesis and hence, cell division.

[00263] While the vast majority of altered cell cycle genes exhibited reduced expression following hsa-let-7 application, a few cell cycle genes were up-regulated under the same conditions, indicating a 2 or 3 effect of hsa-let-7 application. These genes (Table 7) included those encoding CDK inhibitor 2B (CDKN2B), the MAX-interacting protein 1(MXII) - a transcription regalator that antagonizes MYC, and cyclin G2 (CCNG2), which is down-regulated in thyroid papillary carcinoma (Ito et aL, 2003), showing that in tumor cells it has the propensity to act as a tumor antagonist. In let-7-deficient tumor cells, these three genes would likely be down-regulated, which would most likely disable their tumor-suppressing functions.

[00264] Hsa-let-7 addition repressed expression of a number of known and putative tumor suppressor genes (Table 7) such as BRCA1, BRCA2, FANCD2, PLAGLl, E2F6, E2F8, and the cell cycle checkpoint genes CHEKl, BUBI, BUB1B, MAD2L1 and CDC23.

EXAMPLE 5:

[00265] Genes directly targeted by hsa-let-7 may exhibit modified expression prior to 72 hours following hsa-let-7 administration to cells. Therefore, the inventors analyzed gene expression in HepG2 cells harvested at 4, 8, 16, 24, 36, 48, 72, and 128 hours after hsa-let-7 transfection as described in Example 1. Affymetrix U133 plus 2 GeneChips were used in the time course study and processed using Affymetrix MAS
5.0 algorithm as the scaling (value set to 500) and summarization method (Affymetrix Statistical Algorithms Description Document Part Number 701137 Rev 3). Because the time course study was un-replicated, the Wilcoxon Signed Rank test (Wilcoxon, 1945) as implemented in the Affymetrix GCOS 1.4 software, was utilized to determine those genes that were differentially expressed relative to time zero. Those genes that were calculated to be absent in 100% of time points were discarded.

[00266] Within 36 hours of hsa-let-7 transfection, 167 genes were down-regulated and were designated early-repressed genes (Table 8). The early-repressed genes include many of the same cell cycle genes listed in Table 13 above (e.g., CCNA2, CDC25A, CDK8, SKP2, AURKA/STK6) as well as additional genes (e.g., CDC16, CDK6) whose expression levels were repressed early but returned to normal levels by 72 hours. Of the 167 early-repressed genes, 125 genes first appeared down-regulated at or before 16 hours, 32 genes first appeared down-regulated between 16 and hours, and 10 first appeared down-regulated between 24 and 36 hours. Several transcription factors besides E2F6, including ID2, CBFB, ZNF336, SMAD4, SOX9, NRIH4, ARID3A, PLAGL2, YAP1 and GTF21, were among the early repressed genes. It is likely that these genes propagate the let-7 effect to their downstream targets. For example, multiple members of the MCM and RFC DNA synthesis complexes were repressed only at later time points and could be targets of these transcription factors.

Table 8. Early-repressed genes following transfection of HepG2 cells with hsa-let-7b.
Gene S mbol RefSeg Transcript ID
Genes repressed by 16 hours C20orf36 NM 018257 C20orf59 NM 022082 C6ort'96 NM 017909 C9orf64 NM 032307 ELOVLi NM 016031 //1 NM 022821 NM_001518 //1 NM_032999 /// NM_033000 /// NM_033001 /// NM_033003 NM_002211 /// NM033666 /// NM033667 /// NM033668 /// NM033669 OK/SW-c1.56 (TUBB) NM 178014 SARAl NM 020150 NM_003158 /// NM_003600 NM_198433 /// NM_198434 /// NM198435 TIAl NM 022037 /// NM 022173 Genes repressed by 24 hours 2'-PDE NM 177966 C13orf23 NM 025138 /// NM 170719 C14orf46 ---C9orf41 NM 152420 CSNK2Al NM 001895 /// NM 177559 /// NM 177560 DMD NM_000109 /lI NM_004006 /// NM004007 Il/ NM_004009 /// NM_004010 ---Genes repressed by 36 hours NM 001952 /// NM_198256/1/ NM_198257 /11 NM198258NM_198325 OPRS 1 NM 005866 /lI NM 147157 lll NM 147158 /// NM 1 47 1 5 9 111 NM 147160 Pfs2 NM 016095 EXAMPLE 6:

[00267] The 3' untranslated regions (3'UTRs) of let-7 early repressed genes and of genes repressed after 36 hours were examined for the presence of sequences that displayed features of let-7 complementary sites (LCS) in validated let-7 target genes (Johnson et al., 2005; Reinhart et al., 2000; Grosshans et al., 2005; Lin et al., 2003;
Slack et aL, 2000; Vella et al., 2004a; Vella et al., 2004b). Results are shown in Table 9 below.

Table 9. Hsa-let-7 repressed genes with let-7 complementary sites (LCSs) Genes repressed by 16 hours # of Iet-7 LCSs Genes repressed by 24 hours # of Iet-7 LCSs 2'-PDE 5 DMD I
Genes repressed by 36 hours # of let-7 LCSs Genes repressed after 36 hours # of Iet-7 LCSs [00268] At least 25 of the early-repressed genes contained LCSs in their 3'UTRs and likely represent direct let-7 targets. This set includes the cell cycle regulators CDK6, CDC25A, AURKA/STK6, CDCA7, the DNA synthesis regulator RRM2, and the transcription factors CBFB, PLAGL2, E2F6, SOX9, ZNF336, YAP1, GTF2I, and ARID3A. In addition, other cell cycle genes with LCSs in their 3'UTRs were repressed later than 36 hours (E2F5, CDC34, CCNF CCNJ) suggesting that later repressed genes are also direct let-7 targets. The non-LCS containing genes with altered expression upon let-7 addition are likely to be downstream genes indirectly affected by let-7 expression, perhaps as downstream targets of the transcription factors affected directly by let-7.

EXAMPLE 7:

[00269] miRNAs can directly affect mRNA levels of their target genes and will also directly affect protein levels following translational regulation upon binding to target mRNAs. Translational regulation leading to an up or down change in protein expression may lead to changes in activity and expression of downstream gene products and genes that are in turn regulated by those proteins. These regulatory effects would be revealed as changes in the global mRNA expression profile.
The identity and nature of the cellular pathways affected by the regulatory cascade induced by hsa-let-7 expression were determined. Cellular pathway analysis was performed using Ingenuity Pathways Analysis (Ingenuity Systems, Redwood City, CA). The most significantly affected pathways following over-expression of hsa-let7b in A549 and HepG2 cells are shown in Table 10.

Table 10. Significantly affected functional cellular pathways following hsa-let-7b over-expression in A549 and HepG2 cells.
Functional Cellular Pathways Altered by hsa-let 7 Over-Expression A549 HepG2 Amino Acid Metabolism Amino Acid Metabolism Behavior Cancer Cancer Carbohydrate Metabolism Carbohydrate Metabolism Cardiovascular Disease Cardiovascular Disease Cardiovascular System Development and Function Cardiovascular System Development and Function Cell Cycle Cell Cycle Cell Death Cell Death Cell Mo holo Cell Mo holo Cell Si nalin Cell Si lin Cell-To-Cell Signaling and Interaction Cell-To-Cell Si nalin and Interaction Cellular Assembly and Organization Cellular Assembly and Organization Cellular Compromise Cellular Compromise Cellular Development Cellular Development Cellular Function and Maintenance Cellular Function and Maintenance Cellular Growth and Proliferation Cellular Growth and Proliferation Cellular Movement Cellular Movement Cellular Response to Therapeutics Connective Tissue Development and Function Connective Tissue Development and Function Connective Tissue Disorders Connective Tissue Disorders Dermatolo ical Diseases and Conditions Dermatological Diseases and Conditions Developmental Disorder Digestive System Development and Function Digestive System Development and Function DNA Replication, Recombination, and Repair DNA Replication, Recombination, and Repair Drug Metabolism Dru Metabolism Emb onic Development Embryonic Development Endocrine System Development and Function Endocrine System Develo ment and Function Endocrine System Disorders Endocrine System Disorders Free Radical Scavenging Gastrointestinal Disease Gastrointestinal Disease Gene Expression Gene Expression Genetic Disorder Genetic Disorder Hair and Skin Development and Function Hair and Skin Development and Function Hematological Disease Hematological Disease Hematological System Development and Function Hematological System Development and Function Hepatic S stem Development and Function Hepatic System Development and Function Hepatic System Disease He atic System Disease Immune and Lymphatic System Immune and Lymphatic System Development and Development and Function Function Immune Response Immune Response Immunological Disease Immunolo ical Disease Infectious Disease Inflammatory Disease Inflammatory Disease Lipid Metabolism Lipid Metabolism Metabolic Disease Metabolic Disease Molecular Transport Molecular Transport Nervous System Development and Function Nervous System Development and Function Neurological Disease Neurological Disease Nucleic Acid Metabolism Nucleic Acid Metabolism Ophthalmic Disease Organ Development Organ Development Organ Mo holo Organ Mo holo Organismal Development Organismal Development Organismal Functions Organismal Functions Organismal Injury and Abnormalities Organismal Injury and Abnormalities Organismal Survival Post-Translational Modification Protein Synthesis Protein Trafficking Protein Trafficking Renal and Urological Disease Renal and Urolo ical Disease Renal and Urological System Development Renal and Urological System Development and and Function Function Reproductive System Development and Function Reproductive System Development and Function Reproductive System Disease Reproductive System Disease Res irato Disease Res irato Disease Respiratory System Development and Function Respiratory System Development and Function RNA Damage and Repair RNA Post-Transcriptional Modification Skeletal and Muscular Disorders Skeletal and Muscular Disorders Skeletal and Muscular System Skeletal and Muscular System Development and Develo ment and Function Function Small Molecule Biochemistry Small Molecule Biochemistry Tissue Development Tissue Development Tissue Mo holo Tissue Mo holo Tumor Morphology Tumor Mo holog Viral Function Viral Function Viral Infection Visual System Development and Function Vitanzin and Mineral Metabolism Vitamin and Mineral Metabolism [00270] Additional cellular pathway analyses were performed with gene expression data from HepG2 cells, by grouping differentially expressed genes according to their biological functions and using the Gene Ontology (GO) database (Ashburner et al., 2000). The most significantly affected Gene Ontology categories in HepG2 cells are shown in Table 11 (following hsa-let-7 over-expression for 72 hours as described in Example 1) and in Table 12 (following hsa-let-7 over-expression for 4-108 hours as described in Example 5). mRNAs whose expression levels were affected by greater than 2-fold with p-values below 0.05 were identified and classified using Gene Ontology categories. P-values were calculated with hypergeometric tests to determine whether there was a significant enrichment of affected genes in a Gene Ontology category when compared to all genes represented on the arrays.

Table 11. Most significantly affected Gene Ontology categories following hsa-let-7 over ex ression in HepG2 cells for 72 hours.
GO ID GO description P value GO:0006260 DNA replication 5.8E-16 GO:0000087 M hase of mitotic cell cycle 6.2E-13 GO:0000278 Mitotic cell cycle 6.4E-13 GO:0000075 cell cycle checkpoint 1.2E-10 GO:0051301 cell division 8.1E-10 GO:0006270 DNA replication initiation 1.2E-09 GO:0007093 Mitotic checkpoint 2.3E-06 GO:0007051 spindle organization and biogenesis 3.4E-06 Table 12. Most significantly affected Gene Ontology categories following let-7 over expression in He G2 cells over a period of 4 hours to 108 hours.
# of % of genes GO category # of genes in GO
GO ID description altered in category P value genes cate o altered GO:0000278 Mitotic cell cycle 19 192 10 5.5E-08 GO:0051301 cell division 15 135 11 3.1E-07 GO:0000279 M hase 15 165 9 3.8E-06 GO:0007088 regulation of 6 34 18 8.9E-05 mitosis GO:0016126 sterol biosynthesis 5 24 21 1.5E-04 GO:0005525 GTP binding 16 262 6 2.OE-04 GO:0006260 DNA replication 11 138 8 2.2E-04 GO:0051325 Interphase 8 76 11 2.5E-04 [00271] These data demonstrate that hsa-let-7 directly or indirectly affects the expression of many cell cycle-related genes and thus primarily affects cellular functional pathways related to the cell cycle, cell division, and DNA
replication.
Those cellular processes all have integral roles in the development and progression of various cancers.

EXAMPLE 8:

FOR TREATMENT OF CANCERS

[00272] Proliferation and survival pathways are commonly altered in tumors (Hanahan and Weinberg, 2000). The inventors have shown that hsa-let-7 expression directly or indirectly regulates multiple cell proliferation genes. Hsa-let-7 directly regulates a few key cell cycle proto-oncogenes, thus controlling cell proliferation pathways. These data strongly support the assertion that let-7 is a tumor suppressor miRNA.
[00273] A review of the genes and related pathways that are regulated by let-7 indicates that introduction of hsa-let-7 or an anti-hsa-let-7 (anti-miR) into a variety of cancer cell types would likely result in a therapeutic response. Hsa-let-7 targets that have prognostic and/or therapeutic value for the treatment of various malignancies are shown in Table 13.

Table 13. Hsa-let-7 targets having prognostic or therapeutic value for the treatment of various malignancies.
Gene Gene Cellular Cancer Type References Symbol Title Process ATRX ATR-X transcription AML, alpha (Lacayo et al., 2004; Steensma et al., thalassemia 2005; Serrano et al., 2006) AURKA/ aurora chromosomal BC, CRC, PaC, Reiter et al., 2006; Ulisse et al., STK6 kinase A stability OC, GC, SCCHN, 2006; Keen and Taylor, 2004 AURKB/ aurora chromosomal PC, NSCLC, BC, Keen and Taylor, 2004; Chieffi et al., STK12 kinase B stability CRC 2006; Smith et al., 2005 BRCAl BRCA-1 chromosomal BC, OC Wooster and Weber, 2003 stability BRCA2 BRCA-2 chromosomal stability BC, OC Wooster and Weber, 2003 chromosomal AML, SGT, ALL Shigeishi et al., 2006; Grabsch et al., BUB1 BUB1 stability HL, L, CRC, GC ' 2003; Qian et al., 2002; Ru et al., 2002; Cahill et a1.,1998 BUB1B BUBRI chromosomal LC, GC Grabsch et al., 2003; Seike et al., stability 2002 benzodiaz (Hardwick et al., 1999; Sutter et al., epine L, BC, G, CRC, 2002; Han et al., 2003; Kletsas et al., BZRP receptor, apoptosis AC, PC, FS, 2004; Furre et al., 2005; Maaser et peripheral OepC al., 2005; Pretner et al., 2006;
type Vlodavsky and Soustiel, 2007) CCNA2 cyclin A2 cell cycle AML Qian et al., 2002 CCNBI cyclin B 1 cell cycle HCC PC, CRC BC,,CHNLC, Egloff et al., 2006 CCNE2 cyclin E2 cell cycle BC, LC, OC, EC Payton & Coats, 2002; Payton et al., CCNG2 cyclin G2 cell cycle TC, SCCHN Alevizos et al., 2001; Ito et al., 2003 NHL, CRC, (Wolowiec et al., 1999; Egilmez et CDC2 CDK1 cell cycle SCCHN, OepC al., 2001; Chang et al., 2005a;
Hansel et al., 2005) cell CDC20 division cell cycle GC Kim et al., 2005 cycle 20 cell CDC23 division cell cycle CRC Wang et al., 2003 cycle 23 cell HCC, OepC, BC, , , CDC25A division cell cycle CRC NSCLC, O CHN, TC, ~tstjansdottir & Rudolph, cycle 25A NHL
cell CDC6 division cell cycle PC,CeC Murphy et al., 2005; Robles et al., cycle 6 JPO1/CD CRC, OC, LC, CDCA7 CA7 cell cycle GC, EC, AML, Osthus et al., 2005 CML
CDK2 CDK-2 cell cycle OC, CRC, PC Cipriano & Chen, 1998: Marone et al., 1998; Yamamoto et al., 1998 G, GB, GBM, Costello et al., 1997; Lam et al., CDK6 CDK-6 cell cycle MB, B-cell CLL 2000; Hayette et al., 2003; Mendrzyk et al., 2005 CDK Christiansen et al., 2003; Teofili et CDKN2 inhibitor PML, BIdC, al., 2003;
B 2B/p15IN cell cycle ~L, MM, AML le Frere-Belda et al., 2001; Martinez-K4B Delgado et al., 2000; Ng et a1.,1997 CDT1 Cdti chromosomal stability NSCLC Karakaidos et al., 2004 CEBPD C/EBP transcription PC
delta (Yang et al., 2001) CKS1B Cksl cell cycle NSCLC, BC, Inui et al., 2003; Slotky et al., 2005;
CRC Sha iraetal.,2005 CSFI CSF-1 signal HCC LC (Budhu et al., 2006; Uemura et al., transduction ' 2006) (Graff and Zimmer, 2003; Huusko et BC, CRC, NHL, al., 2004; Nakada et al., 2004; Wu et EIF4E eIF-4e translation NB, CHN, LXC, al., 2004; Jubb et al., 2005; Guo et B1dC, PC, GC al., 2006; Kokko et al., 2006; Wu et al., 2006; Davalos et al., 2007) (Huusko et al., 2004; Nakada et al., EPH 2004; Wu et al., 2004; Jubb et al., EPHB2 receptor signal PC, GC, CRC, 2005; Guo et al., 2006; Kokko et al., B2 transduction OC, G, BC 2006; Wu et al., 2006; Davalos et al., 2007) (Lemoine et al., 1992; Rajkumar et signal PC, BC, pilocytic al., 1996; Leng et al., 1997; Maurer ERBB3 HER-3 AC, GC, CRC, et al., 1998; Kobayashi et al., 2003;
transduction OC, B1dC Koumakpayi et al., 2006; Xue et al., 2006) fatty acid fat OC, BC, BIdC, (Ye et al., 2000; Camassei et al., FASN CeC, PC, RB, 2003; Menendez et a1., 2004;
synthase metabolism CRC Kuhajda, 2006) FGFBP 1 FGF-BP signal SCCHN, BC, (Abuharbeid et al., 2006; Tassi et al., transduction CRC, PC, PaC 2006) FGFR4 FGF signal TC BC OC PaC (Jaakkola et al., 1993; Shah et al., rec tor-4 transduction ' ' ' 2002; Ezzat et al., 2005) FH fumarase sugar RCC, LM (Eng et al., 2003) metabolism GMNN Geminin DNA CeC Shetty et al., 2005; Bravou et al., replication CRC, BC, 2005; Wohlschle el et al., 2002 IGFBPI IGFBP-1 signal BC, CRC (Firth and Baxter, 2002) transduction BC, CRC, signal ' (Akiba et al., 2001; Sparmann and IL8 IL-8 transduction H CLC, PC, Bar-Sagi, 2004) Wewer et al., 1997; Aplin et al., integrin BC, CeC, HCC, 1996; Begum et al., 1995; Rabinovitz ITGA6 alpha-6 cell adhesion LC et al., 1995; Mariani Costantini et al., JUN c-Jun transcription HL, HCC (Eferl et al., 2003; Weiss and Bohniann, 2004) (Bossy-Wetzel et al., 1992; Mathas L, CML, HCC, et al., 2002; Mao et al., 2003; Yang JUNB Jun B transcription TCL, HL, FS et al., 2003; Passegue et al., 2004;
Chang et al., 2005b; Liu et al., 2006;
Ott et al., 2007) lipoma LHFP HMGIC transcription Li (Petit et al., 1999) fusion partner MCAM MCAM cell adhesion M, AS, KS, LMS McGary et a1., 2002 SPRC, HCC, GC, signal SCCHN, OS, MET c-Met transduction RMS, GB, BC, (Boccaccio and Comoglio, 2006) M, CRC, GI, PaC, PC, OC
major multi drug AML, CML, MVP vault ALL, OC, BC, M, (Mossink et al., 2003) protein resistance OS, NB, NSCLC
Max- Ariyanayagam-Baksh et al., 2003;
MXII interacting transcription M, PC, GB Prochownik et al., 1998; Wechsler et protein 1 al., 1997 MYBLI A-Myb transcription BL (Golay et al., 1996) BC, NSCLC, PC, (Tanner et al., 2000; Bar-Shira et al., MYBL2 Myb L2 transcription OC 2002; Borczuk et al., 2003; Ginestier et al., 2006) M, TC, MM, Demunter et al., 2001; Oyama et al., signal CRC, Shi et al., 1991; Paquette, et NRAS N-Ras transduction , AML, BC, al., 1990; Neri et al, 1989; Gerosa et GC, GB al., 1989; Bos, 1988 P8 P8 transcription BC, TC, PaC (Ree et al., 1999; Su et al., 2001; Ito et al., 2005) (Chen et al., 2003; Jansen et al., PDCD4 Pdcd-4 apoptosis G, HCC, L, RCC 2004; Zhang et al., 2006; Gao et al., 2007) NSCLC, OrpC, OepC, GC, M, PLKI polo-like chromosomal BC, OC, EC, (Strebhardt and Ullrich, 2006) kinase 1 stability CRC, GB, PapC, PaC, PC, HB, NHL
B1dC, PC, EC, (Weichert et al., 2003; Jiang et al., PRKCA PKC signal BC, CRC, HCC, 2004; Lahn and Sundell, 2004;
alpha transduction M, GC, OC Koivunen et al., 2006) RASSF2 RASSF2 signal GC, CRC, OC (Akino et al., 2005; Endoh et al., transduction 2005; Lambros et al., 2005) SIVA CD27 apoptosis BC (Chu et al., 2005) binding Einama et al., 2006; Traub et al., proteasomal PaC, OC, BC, 2006; Sui et al., 2006; Huang et al., SKP2 SKP-2 degradation MFS, GB, EC, 2006; Saigusa et al., 2005; Shibahara NSCLC, PC et al., 2005; Kamata et al., 2005;
Takanami, 2005 PaC, CRC, BC, SMAD4 SMAD-4 signal SCCHN, AML, Miyaki and Kuroki, 2003 transduction GC, HCC, OC, szc TACC3 TACC3 cell cycle OC, NSCLC (Lauffart et al., 2005; Jung et al., 2006) E2F (Halaban et al., 2000; Wang et al., TFDPi dimerizati cell cycle M, HCC, NHL 2001; Chan et al., 2002; Yasui et al., on artner 2002) TGF beta signal CeC high grade Soufla et al., 2005; Woszczyk et al., TGFBR3 receptor transduction NHL, BC 2004; Bandyopadhyay et al., 2002;
III ' ' Venkatasubbarao et al., 2000 TNFSFl TRAIL apoptosis CRC, G, LC, PC, (Fesik, 2005) 0 multiple ML
(Caselitz et al., 1983; Stark et al., 1984; Ben-Ze`ev and Raz, 1985;
HCC, M, L, BC, Churg,1985; Upton et al., 1986;
adhesion and PC, CeC, CRC, Ferrari et al., 1990; Sommers et al., VIM vimentin RCC, SCCHN, 1992; Gilles et al., 1996; Rutka el al., migration AC, CLL, MT, 1999; Islam et al., 2000; Khoury et LC al., 2002; Singh et al., 2003; Hu et al., 2004; Fesik, 2005; Mclnroy and Maatta, 2007; Ngan et al., 2007) Abbreviations: AC, astrocytoma; ALL, acute lymphocytic leukemia; alpha thalassemia, alpha thalassemia; AML, acute myeloid leukemia; AS, angiosarcoma; BC, breast carcinoma; BL, Burkitt's lymphoma; B1dC, bladder carcinoma; CeC, cervical carcinoma; CHN, carcinoma of the head and neck;
CLL, chronic lymphocytic leukemia; CML, chronic myeloblastic leukemia; CRC, colorectal carcinoma; EC, endometrial carcinoma; FS, fibrosarcoma; G, glioma; GB, glioblastoma; GBM, glioblastoma multiforme; GC, gastric carcinoma; GI, gastrinoma; HB, hepatoblastoma; HCC, hepatocellular carcinoma; HL, Hodgkin lymphoma; KS, Kaposi's sarcoma; L, leukemia; LC, lung carcinoma; Li, lipoma; LM, leiomyoma; LMS, leiomyosarcoma; LXC, larynx carcinoma; M, melanoma; MB, medulloblastoma; MFS, myxofibrosarcoma; ML, myeloid leukemia;
MM, multiple myeloma; MT, mesothelioma; NB, neuroblastoma; NHL, non-Hodgkin lymphoma;
NSCLC, non-small cell lung carcinoma; OC, ovarian carcinoma; OecP, oesophageal carcinoma; OrpC, oropharyngeal carcinoma; OS, osteosarcoma; PaC, pancreatic carcinoma; PapC, papillary carcinoma; PC, prostate carcinoma; PML, promyelocytic leukemia; RB, retinoblastoma; RCC, renal cell carcinoma; RMS, rhabdomyosarcoma; SCCHN, squamous cell carcinoma of the head and neck; SGT, salivary gland tumor; SIC, small intestinal carcinoma; SPRC, sporadic papillary renal carcinoma; TC, thyroid carcinoma; TCL, T-cell leukemia; UC, urothelial carcinoma [00274] These targets are critical regulators of angiogenesis, chromosomal stability, cell adhesion, invasion, cell cycle progression, transcription, DNA
replication and intracellular signal transduction. Hsa-let-7 affects intracellular signaling at various layers and controls the expression of secretory proteins, transmembrane growth factor receptors as well as cytoplasmic signaling molecules Among secretory proteins are fibroblast growth factor binding protein (FGFBPI), insulin growth factor binding protein (IGFBP1) and the inflammatory chemokine interleukin 8(IL8). FGFBP I is a secretory protein stored in an inactive form on heparin sulfate proteoglycans in the extracellular matrix (Tassi et al., 2001;
Abuharbeid et al., 2006). It has high affinity for FGF-1 and FGF-2 and functions as chaperone to mobilize locally stored FGF. Thus, FGFBPI is a positive regulator of FGFs enhancing FGF signaling and angiogenesis (Tassi et al. 2001). FGFBPI
expression is highly tissue specific and absent in most normal adult tissues.
Yet, FGFBP l is overexpressed in various types of cancer, including cancers of the breast, colon and prostate (Abuharbeid et al. 2006). High FGFBPI expression is associated with early stages of tumor development, contributing to tumor angiogenesis.
Similarly, IL-8 is frequently upregulated in various cancers and correlates with tumor vascularization, metastasis and poor prognosis (Sparmann et al., 2004;
Rosenkilde et al., 2004).

[00275) Membrane-associated proteins regulated by hsa-let-7 are Met (MET), transforming growth factor-beta receptor 3 (TGFBR3), ERBB3 and the Ras association domain family protein 2 (RASSF2). RASSF2 is a tumor suppressor candidate that is frequently downregulated in lung tumor cell lines (Vos et al., 2003).
RASSF2 interacts with K-Ras and promotes cell cycle arrest and apoptosis. Met acts as the receptor for hepatocyte growth factor (HGF) and was isolated as an oncogene from a chemically transformed human cell line (Cooper et al., 1984; Dean et al., 1985). Met activating mutations are found in sporadic papillary renal cancer, childhood hepatocellular carcinoma and gastric cancer (Danilkovitch-Miagkova et al., 2002). These somatic mutations are associated with increased aggressiveness and extensive metastases in various carcinomas. In several other cancer types, autocrine and paracrine mechanisms lead to an activation of Met signaling. The most frequent mechanism of Met activation, however, is overexpression which occurs in colorectal cancer, hepatocellular carcinoma, gastrinomas as well as carcinomas of the stomach, pancreas, prostate, ovary and breast (Boccaccio et al., 2006). Met is overexpression correlates with a metastatic tumor phenotype and poor prognosis (Birchmeier et al., 2003). ERBB3 (also known as HER-3) belongs to the protein family of EGFRs and is a homolog of the avian v-Erb oncoprotein (Hynes et al., 2005). In contrast to EGFR, ERBB3 transmits mitotic signals only when complexed as a heterodimer with other EGFR members such as ERBB2. Various cancer types show increased levels of ERBB3 and consequently an activation of the EGFR pathway (Table 13). TGFBR-3 is a putative tumor suppressor transmitting signals in response to TGF-beta.
Central role of TGF-beta is inhibition of cellular growth of numerous cell types, such as epithelial, endothelial, hematopoietic neural and mesenchymal cells. TGFBR-3, also referred to as beta-glycan, binds all three TGF-beta isoforms with high affinity.
TGFBR-3 associates with TGFBR-2 to signal to downstream effector molecules (Blobe et al., 2001). TGFBR-3 is frequently downregulated in multiple cancer types (Hishikawa et al., 1999; Lin et al., 2005). PRKCA, a cytosolic signaling molecule, belongs to a family of serine-threonine kinases that are activated in response to signaling induced by receptor tyrosine kinases. Functional studies have suggested that PKCs play a role in carcinogenesis and maintenance of the malignant phenotype (Karakaidos et al., 2004; Koivunen et al., 2006). PRKCA is overexpressed in endometrial, prostate and high grade urinary bladder carcinomas (Koivunen et al.
2006). PRKCA activity is linked to increased motility and invasion of cancer cells, a phenotype that can be reversed by PRKCA inhibition (Koivunen et al., 2004).

[00276] Another class of genes and their corresponding proteins that are regulated by hsa-let-7, functions in the progression of the cell cycle. Some of these proteins are critical in the transition through G1 and S phases, such as cyclins A2 and E2 (CCNA2, CCNE2), cyclin dependent kinases 2 and 6 (CDK2, CDK6), the CDK
inhibitor CDKN2B, CDC25A and cell division cycle 6 (CDC6). Others are required for progressing through the G2/M spindle checkpoint and proper segregation of sister chromatids during mitosis to maintain chromosomal stability. These include aurora kinases A and B (AURKA, a.k.a. STK6; AURKB, a.k.a. STK12), breast cancer 1 and 2(BRCA1; BRCA2), budding uninhibited by benzimidazoles 1(BUB1), budding uninhibited by benzimidazoles I beta (BUB1B), polo-like kinase 1(PLK1), cyclin dependent kinase 1(CDKl, a.k.a. CDC2), cyclin B1, and cell division cycle 20 and 23 (CDC20, CDC23, a.k.a. anaphase promoting complex subunit 8). Most of these transcripts are regulated in a manner that suggests that hsa-let-7 blocks cell cycle progression. All of these targets also have an evident role in carcinogenesis.
For instance, the tumor suppressor proteins BRCAI and BRCA2, as well as the growth-promoting aurora kinases A and B show deregulated expression in a various solid tumors, e.g. carcinomas of the breast, ovary, thyroid gland, lung, prostate and colorectum (Chieffi et al., 2006; Keen et al., 2004; Smith et al., 2005;
Ulisse et al., 2006; Wooster et al., 2003; Turner et al., 2004). Aurora kinases are preferred drug targets in the pharmaceutical industry. PLKI (also referred to as serine-threonine protein kinase 13; STPK13) is a protein kinase that regulates mitotic spindle function to maintain chromosomal stability (Strebhardt et al., 2006). PLKI expression is tightly regulated during the cell cycle and peaks in M phase. PLKI is inherently oncogenic and directly inhibits the tumor suppressor function of p53 (Ando et al., 2004). Overexpression of PLK1 induces a polynucleated phenotype and cellular transformation of NIH3T3 cells (Mundt et al., 1997; Smith et al., 1997).
Likewise, PLK1 shows increased expression levels in most solid tumors, including carcinomas of the breast, colon, lung, stomach and prostate (Table 13). PLKl overexpression is associated with disease progression and - when depleted - induces apoptosis in cancer cells (Strebhardt et al. 2006; Liu et al., 2003). Currently, PLK1 is being tested as a target of various small molecule inhibitors for future therapeutic intervention (Strebhardt et al. 2006). CDC6 is regulated in response to mitogenic signals through transcriptional control mechanisms involving E2F proteins and is required for initiation of DNA replication in mammalian cells. CDC6 is overexpressed in various human cancers and has inherent oncogenic potential (Karakaidos et al. 2004;
Gonzalez et al., 2006; Murphy et al., 2005; Semple et al., 2004). Cyclins are co-factors of cyclin-dependent kinases (CDKs) (Malumbres et al., 2001). The expression of cyclins is tightly controlled during the cell cycle to govern the activity of individual CDKs. Cyclin A2 and cyclin E2 associate with CDK2 during S phase; cyclin D1 is the predominant co-factor of CDK4/6 in G 1 phase. Since most cyclins are promoters of cell growth, cyclins - such as cyclin A2, B1 or E2 - are frequently expressed at high levels in various tumor types (Egloff et al., 2006; Payton et al., 2002;
Payton et al., 2002; Qian et al., 2002). CDK6 forms active complexes with D-type cyclins, including D1, D2 and D3. The primary function of CDK2 and CDK6 is to inactivate members of the retinoblastoma protein family. CDK2 and CDK6 are overexpressed in nu.merous cancers and are currently being explored as a potential cancer drug targets (Malumbres et al. 2001; Cipriano et al., 1998; Costello et al., 1997;
Hayette et al., 2003; Lam et al., 2000; Marone et al., 1998; Mendrzyk et al., 2005;Yamamoto et al., 1998). CDK1 (CDC2) is a catalytic subunit of a protein kinase complex, called the M-phase promoting factor that induces entry into mitosis and is universal among eukaryotes. Activation of CDKl requires binding to B cyclins and dephosphorylation by CDC25. Similar to other CDKs, CDK1 is expressed at increased levels in various cancers (Table 13). The CDC25 protein phosphatase family plays a critical role in activating cyclin-dependent kinases (CDKs) via dephosphorylation of conserved threonine 15 and tyrosine 15 inhibitory phosphorylation sites. While CDC25C is primarily responsible for activating CDK1 to overcome G2/M checkpoint and allow mitotic entry, the primary substrate of CDC25A is CDK2 and CDK6 which - when active - allows progression through the Gl/S and intra-S checkpoints (Kristjansdottir et al., 2004). CDC25A is frequently amplified and overexpressed in human cancers, including cancers of the breast, lung, rectum and brain (Kristjansdottir et al. 2004).
Other molecules regulated by hsa-let-7 that indirectly control cell cycle progression are SKP2 and PDCD4 (programmed cell death 4). Skp2 is a component of the multi-subunit E3 ubiquitin ligase complex that ear-marks proteins for proteasomal degradation. A well characterized target is the CDK inhibitor p27 which offers an explanation for the cell cycle promoting activity of Skp2 (Carrano et al., 1999). Skp2 is inherently oncogenic and shows elevated levels in various cancer types (Gstaiger et al., 2001; Einama et al., 2006; Kamata et al., 2005; Saigusa et al., 2005).
PDCD4 is a tumor suppressor that is induced in response to apoptosis in normal cells. The growth inhibitory properties of PDCD4 are due to PDCD4-mediated inhibition of the c-Jun proto-oncoprotein, inhibition of cap-dependent mRNA translation and activation of the p2lWafl/Cipl CDK inhibitor (Bitomsky et al., 2004; Goke et al., 2004; Yang et al., 2003). PDCD4 frequently shows reduced or lost expression in various human malignancies, such as gliomas, hepatocellular carcinomas, lung and renal cell carcinomas (Gao et al., 2007; Jansen et al., 2004; Zhang et al., 2006).
Expression of PDCD4 interferes with skin carcinogenesis in a mouse model and suppresses growth of human colon carcinoma cells (Jansen et al., 2005; Yang et al., 2006). Loss of PDCD4 also correlates with lung tumor progression (Chen et al., 2003).

[00277] Hsa-let-7 also regulates vimentin (VIM) and fatty acid synthase (FASN).
Vimentin is the major intermediate filament protein of inesenchymal cells and governs proteins that are critical in attachment, migration and cell signaling.
Vimentin has key roles in adhesion by regulating integrin functions. Vimentin is often expressed at elevated levels in human malignancies which correlates with invasiveness and poor survival (Caselitz et al., 1983; Churg 1985; Upton et al., 1986;
Gilles et al., 1996; Islam et al., 2000; Ngan et al., 2007; Sommers et al., 1992; Singh et al., 2003). Downregulation of vimentin expression by RNA interference inhibits carcinoma cell migration and adhesion (McInroy et al., 2007). FASN, often referred to as FAS, is the sole protein in the human genome capable of the reductive de novo synthesis of long-chain fatty acids from acetyl-CoA, malonyl-CoA, and nicotinamide adenine dinucleotide phosphate (NADPH) (Kuhajda 2006). FAS is elevated and active in various cancer cells. Since fatty acid synthesis expends energy, FAS
expression might confer some survival or growth advantage to human cancer cells.
FAS expression correlates with poor prognosis of patients with carcinomas of the lung, breast, prostate, skin and soft-tissue sarcomas and is predictive for recurrence of prostate cancer (Kuhajda 2006).

[00278] In summary, let-7 controls a variety of cancer genes that play key roles in the development or progression of the disease. These targets are frequently deregulated in human cancer. Based on this review of the genes and related pathways that are regulated by hsa-let-7, introduction of hsa-let-7 or inhibitory anti-hsa-let-7 into a variety of cancer cell types would likely result in a therapeutic response EXAMPLE 10:

LUNG CANCER CELLS

[00279] The inventors have previously demonstrated that hsa-let-7 is involved in the regulation of numerous cell activities that represent intervention points for cancer therapy and for therapy of other diseases and disorders (U.S. Patent Applications serial number 11/141,707 filed May 31, 2005 and serial number 11/273,640 filed November 14, 2005). For example, depending on the cell type, overexpression of hsa-let-7 may increase or decrease the proliferation and/or viability of certain normal or cancerous cell lines, and overexpression of let-7 in cells may also induce a significant shift toward or away from a specific stage of the cell cycle.

[00280] The development of effective therapeutic regimens requires evidence that demonstrates efficacy and utility of the therapeutic in various cancer models and multiple cancer cell lines that represent the same disease. The inventors assessed the therapeutic effect of hsa-let-7 for lung cancer by measuring cellular proliferation using six non-small cell lung cancer (NSCLC) cell lines, including cells derived from lung adenocareinoma (A549, H838, Calu-3, HCC2935), cells derived from lung squamous cell carcinoma (H226), and cells derived from lung adenosquamous cell carcinoma (1-1596). The inventors also measured proliferation of cells derived from lung large cell carcinoma (H460). Cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). Synthetic hsa-let-7b, hsa-let-7c, or hsa-let-7g (Pre-miRTM-hsa-let-7, Ambion cat. no. AM17100) or negative control (NC) miRNA (Pre-miRTM microRNA Precursor Molecule-Negative Control #2; Ambion cat. no. AM17111) was delivered via lipid-based transfection into A549, H838, Calu-3, HCC2935, and H460 cells and via electroporation into H226 cells. Lipid-based reverse transfections were carried out in triplicate according to a published protocol (Ovcharenko et al., 2005) and the following parameters: 5000-12000 cells per well, 0.1-0.2 gl LipofectamineTM 2000 (cat. no. 11668-019, Invitrogen Corp., Carlsbad, CA, USA) in 20 l OptiMEM (Invitrogen), 30 nM final concentration of miRNA in 100 l. A549, H838, H460, H596 and HCC2935 cells were harvested 72 hours post transfection to evaluate cellular proliferation; Calu-3 cells were analyzed days post transfection. Proliferation assays were performed using Alamar Blue (Invitrogen) following the manufacturer's instructions. As a control for inhibition of cellular proliferation, siRNA against the motor protein kinesin 11, also known as Eg5, was used. Eg5 is essential for cellular survival of most eukaryotic cells and a lack thereof leads to reduced cell proliferation and cell death (Weil et al., 2002). siEg5 was used in lipid-based transfection following the same experimental parameters that apply to miRNA. The inventors also used a topoisomerase II inhibitor, etoposide, at a final concentration of 10 M and 50 M as an internal standard for the potency of miRNAs. Etoposide is an FDA-approved topoisomerase II inhibitor in the treatment of lung cancer. IC50 values for various lung cancer cells have been reported to range between <1-25 M for SCLC and NSCLC cells (Ohsaki et al., 1992; Tsai et al., 1993). Percent (%) proliferation values from the Alamar Blue assay were normalized to values from cells treated with negative control miRNA (NC). Percent proliferation of hsa-let-7 treated cells relative to cells treated with negative control miRNA (100%) is shown below in Table 14 and in FIG. 1.

[002811 Delivery of hsa-let-7b, hsa-let-7c or hsa-let-7g inhibits cellular proliferation of lung cancer cells A549, H838, Calu-3, HCC2935, H596, and H460 (Table 14 and FIG. 1). The inhibitory activity of the three let-7 members, hsa-let-7b, hsa-let-7c, and hsa-let-7g, were similar in all cell lines tested, suggesting a redundant role for these miRNAs. On average, hsa-let-7 inhibits cellular proliferation by 26%
(Table 14 and FIG. 1). Hsa-let-7b, hsa-let-7c and hsa-let-7g have maximal inhibitory activity in H460 cells, reducing proliferation by 68%, 37%, and 43%, respectively.
The growth-inhibitory activity of hsa-let-7 is comparable to that of etoposide at concentrations >10 gM. Since hsa-let-7 induces a therapeutic response in all lung cancer cells tested, hsa-let-7 may provide therapeutic benefit to patients with lung cancer and other malignancies.

[00282] The inventors determined sensitivity and specificity of hsa-let-7 by administering hsa-let-7b or negative control miRNA to H460 cells at increasing concentrations, ranging from 0 pM to 3000 pM (Table 15 and FIG. 2). Delivery of miRNA and assessment of cellular proliferation were done as described above.
Proliferation values from the Alamar Blue assay were normalized to values obtained from mock-transfected cells (0 pM = 100% proliferation). Increasing amounts of negative control miRNA (NC) had no effect on cellular proliferation of H460 cells (Table 15 and FIG. 2). In contrast, the growth-inhibitory phenotype of hsa-let-7b is dose-dependent and correlates with increasing amounts of hsa-let-7b (Table 15 and FIG. 2). Hsa-let-7b induces a specific therapeutic response at concentrations as low as 300 pM.

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d~~ x U x WO 2008/073922 PCT[US2007/087037 Table 15. Dose-dependent inhibition of cellular proliferation of H460 lung cancer cell lines by hsa-let-7b. Values are normalized to values obtained from mock-transfected cells (0 pM miRNA). NC, negative control miRNA; %SD, standard deviation.

miRNA
hsa-let-7b NC
Concentration % % % %
[ M] proliferation SD proliferation SD
0 100.00 8.84 100.00 8.84 3 108.28 0.92 107.60 0.79 30 101.96 1.14 108.04 1.46 300 74.14 1.32 106.99 4.74 3 000 27.76 1.54 91.41 2.14 [00283] To evaluate the inhibitory phenotype of hsa-let-7 over an extended period of time, the inventors conducted growth curve experiments in the presence of hsa-let-7 for up to 21 days with H226 cells. Since in vitro transfections of naked interfering RNAs, such as synthetic miRNA, are transient by nature and compromised by the dilution of the oligonucleotide during ongoing cell divisions, hsa-let-7b was administered at multiple time points via electroporation (Bartlett et al., 2006, Bartlett et al., 2007). Equal numbers of H226 cells were electroporated with 1.6 M
synthetic hsa-let-7b (Pre-miRTM-hsa-let-7b, Ambion cat. no. AM17100) or negative control miRNA (Pre-miRTM microRNA Precursor Molecule-Negative Control #2; Ambion cat. no. AM17111) using a Gene Pulser Xce11TM electroporation system (BioRad Laboratories, Inc.; Hercules, CA, USA) (day 0) with the following settings: >0-20 x 106 cells with 5 g hsa-let-7b in 200 l OptiMEM (Invitrogen) (1.6 M miRNA), square wave pulse at 250 V for 5 ms. Electroporated cells (106) were seeded and propagated in regular growth medium. On days 6, 10, and 17, cells were repeatedly harvested, counted, and electroporated with 1.6 gM hsa-let-7b or negative control miRNA. After electroporation on day 6, all cells were re-seeded onto culture dishes.
On days 10 and 17, 50% (cells treated with hsa-let-7b) or 25% (cells treated with negative control miRNA) of the actual cell count was electroporated and propagated to accommodate exponential cell growth. Cell counts from these electroporation events were extrapolated and plotted on a linear scale.

[00284] As shown in FIG. 3, four equal doses of synthetic hsa-let-7b miRNA
over 21 days in 4-7 day intervals resulted in an approximate 85% inhibition of H226 cell growth relative to cells that received negative control miRNA. The data suggest that multiple administrations of hsa-let-7b enhance the therapeutic effect of let-7 miRNA
and reinforce previous data, indicating the therapeutic potential of hsa-let-7 miRNA.

EXAMPLE 11:
HSA-LET-7. IN COMBINATION WITH SPECIFIC HUMAN MICRO-RNAS, SYNERGISTICALLY INHIBITS PROLIFERATION OF LUNG CANCER
CELL LINES

[00285] miRNAs function in multiple pathways controlling multiple cellular processes. Cancer cells frequently show aberrations in several different pathways, which determine their oncogenic properties. Therefore, administration of multiple miRNAs to cancer patients may result in a superior therapeutic benefit over administration of a single miRNA. The inventors assessed the efficacy of pair-wise miRNA combinations, administering hsa-let-7b, hsa-let-7c or hsa-let-7g concurrently with either hsa-miR-34a, hsa-miR-124a, hsa-miR-126 or hsa-miR-147 (Pre-miRTM
miRNA, Ambion cat. no. AM17100). H460 lung cancer cells were transiently reverse-transfected in triplicates with each miRNA at a final concentration of 300 pM, resulting in 600 pM of total oligonucleotide. For negative controls, 600 pM of Pre-miRTM microRNA Precursor Molecule-Negative Control #2 (Ambion cat. no.
AM17111) were used. To correlate the effect of various combinations with the effect of the sole miRNA, each miRNA at 300 pM was also combined with 300 pM negative control miRNA. Reverse transfections used the following parameters: 7,000 cells per 96 well, 0.15 l LipofectamineTM 2000 (Invitrogen) in 20 l OptiMEM
(Invitrogen), 100 l total transfection volume. As an internal control for the potency of miRNA, etoposide was added at 10 M and 50 M to mock-transfected cells, 24 hours after transfection for the following 48 hours. Cells were harvested 72 hours after transfection and subjected to Alamar Blue assays (Invitrogen). Percent proliferation values from the Alamar Blue assay were normalized to those obtained from cells treated with 600 pM negative control miRNA. Data are expressed as %
proliferation relative to negative control miRNA-treated cells (Table 16.).

[00286] Transfection of 300 pM hsa-let-7 reduces proliferation of H460 cells by 30.57% (Table 16 and FIG. 4). Additive activity of pair-wise combinations (e.g. hsa-let-7 plus hsa-let-7g) is defined as an activity that is greater than the sole activity of each miRNA (e.g., the activity of hsa-let-7b plus hsa-miR-126 is greater than that observed for hsa-let-7b plus NC and the activity of hsa-let-7b plus hsa-miR-126 is greater than that observed for hsa-miR-126 plus NC). Synergistic activity of pair-wise combinations is defined as an activity that is greater than the sum of the sole activity of each miRNA (e.g., the activity of hsa-let-7b plus hsa-miR-34a is greater than that observed for the sum of the activity of hsa-let-7b plus NC and the activity of hsa-miR-34a plus NC). The data indicate that hsa-let-7c or hsa-let-7g combined with either hsa-miR-34a, hsa-miR-124a, hsa-miR-126, hsa-miR-147, or hsa-let-7b results in synergistic activity (Table 16 and FIG. 4). Therefore, administering combinations of hsa-let-7 with other miRNAs to cancer patients may induce a superior therapeutic response in the treatment of lung cancer. The combinatorial use of miRNAs represents a potentially useful therapy for cancer and other diseases.

Table 16. Cellular proliferation of H4601ung cancer cells in the presence of pair-wise hsa-let-7 miRNA combinations. Values are normalized to values obtained from cells transfected with 600 pM negative control (NC) miRNA. SD, standard deviation S; synergistic effect; A, additive effect.

miRNA [300 Ml + miRNA [300 pMl Proliferation SD Effect NC + NC 100.00 1.45 NC + miR-34a 99.58 1.66 NC + miR-124a 69.43 1.38 NC + miR-126 89.46 2.27 NC + miR-147 76.97 1.46 NC + let-7b 74.92 3.38 NC + let-7c 86.74 2.28 NC + let-7 91.41 3.26 miR-34a + let-7b 64.85 3.50 S
miR-34a + let-7c 76.41 3.81 S
miR-34a + let-7 73.83 2.85 S
miR-124a + let-7b 39.77 7.61 S
miR-124a + let-7c 37.35 3.08 S
miR-124a + let-7 35.15 0.84 S
miR-126 + let-7b 68.76 5.89 A
miR-126 + let-7c 57.03 5.15 S
miR-126 + let-7 61.89 3.27 S
miR-147 + let-7b 56.55 3.85 A
miR-147 + let-7c 60.74 0.60 S
miR-147 + let-7 56.19 2.95 S
let-7b + let-7c 48.07 3.75 S
let-7b + let-7g 43.19 1.71 S

let-7c + let-7g 59.85 6.70 S
Etoposide (10 M) 20.19 1.89 Etoposide (50 M) 14.94 0.31 EXAMPLE 12:

LUNG CANCER CELLS IN MICE

[00287] The inventors assessed the growth-inhibitory activity of hsa-let-7b in human lung cancer xenografts grown in immunodeficient mice. Hsa-let-7b was delivered into A549 lung cancer cells via electroporation using the Gene Pulser Xcel1TM (BioRad) with the following settings: 15 x 106 cells with 5 g miRNA
in 200 l OptiMEM, square wave pulse at 150 V for 10 ms. As a negative control, A549 cells were electroporated with negative control (NC) miRNA (Pre-miRTM microRNA
Precursor Molecule-Negative Control #2; Ambion cat. no. AM17111) as described above. To assess the anti-oncogenic activity of hsa-let-7b, a group of 4 animals was injected with A459 cells. Electroporated cells (5 x 106) were mixed with BD
MatrigelTM, (BD Biosciences; San Jose, CA, USA; cat. no. 356237) in a 1:1 ratio and injected subcutaneously into the flank of NOD/SCID mice (Charles River Laboratories, Inc.; Wilmington, MA, USA) (day 0). NC miRNA-treated cells were injected into the opposite flank of the same animal to control for animal-to-animal variability. Once tumors reached a measurable size (day 12), the length and width of tumors were determined daily or every other day for up to 18 days. Tumor volumes were calculated using the formula, Volume = length X width X width / 2, in which the length is greater than the width. Tumor volumes derived from NC-treated cells and hsa-let-7b-treated cells were averaged and plotted over time (FIG. 5). Data points with p values <0.05, indicating statistical significance, are indicated by asterisks (days 12-19).

[00288] Administration of hsa-let-7b into the A549 lung cancer xenografts inhibited tumor growth in vivo (FIG. 5). Cancer cells that received negative control miRNA developed tumors more rapidly than cells treated with hsa-let-7b.
Administration of hsa-let-7b into A549 cells suppressed and delayed the onset of tumor growth.

[00289] These data suggest that hsa-let-7 represents a particularly useful candidate in the treatment of lung cancer and potentially other diseases.

REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

U.S. Patent 4,337,063 U.S. Patent 4,404,289 U.S. Patent 4,405,711 U.S. Patent 4,659,774 U.S. Patent 4,659,774 U.S. Patent 4,682,195 U.S. Patent 4,683,202 U.S. Patent 4,704,362 U.S. Patent 4,816,571 U.S. Patent 4,816,571 U.S. Patent 4,959,463 U.S. Patent 4,959,463 U.S. Patent 5,141,813 U.S. Patent 5,143,854 U.S. Patent 5,202,231 U.S. Patent 5,214,136 U.S. Patent 5,221,619 U.S. Patent 5,223,618 U.S. Patent 5,242,974 U.S. Patent 5,264,566 U.S. Patent 5,268,486 U.S. Patent 5,288,644 U.S. Patent 5,324,633 U.S. Patent 5,378,825 U.S. Patent 5,384,261 U.S. Patent 5,405,783 U.S. Patent 5,412,087 U.S. Patent 5,424,186 U.S. Patent 5,428,148 U.S. Patent 5,429,807 U.S. Patent 5,432,049 U.S. Patent 5,436,327 U.S. Patent 5,445,934 U.S. Patent 5,446,137 U.S. Patent 5,466,786 U.S. Patent 5,468,613 U.S. Patent 5,470,710 U.S. Patent 5,470,967 U.S. Patent 5,472,672 U.S. Patent 5,480,980 U.S. Patent 5,492,806 U.S. Patent 5,503,980 U.S. Patent 5,510,270 U.S. Patent 5,525,464 U.S. Patent 5,527,681 U.S. Patent 5,529,756 U.S. Patent 5,532,128 U.S. Patent 5,545,531 U.S. Patent 5,547,839 U.S. Patent 5,554,501 U.S. Patent 5,554,744 U.S. Patent 5,556,752 U.S. Patent 5,561,071 U.S. Patent 5,571,639 U.S. Patent 5,574,146 U.S. Patent 5,580,726 U.S. Patent 5,580,732 U.S. Patent 5,583,013 U.S. Patent 5,593,839 U.S. Patent 5,599,672 U.S. Patent 5,599,695 U.S. Patent 5,602,240 U.S. Patent 5,602,244 U.S. Patent 5,610,289 U.S. Patent 5,610;287 U.S. Patent 5,614,617 U.S. Patent 5,623,070 U.S. Patent 5,624,711 U.S. Patent 5,631,134 U.S. Patent 5,637,683 U.S. Patent 5,639,603 U.S. Patent 5,645,897 U.S. Patent 5,652,099 U.S. Patent 5,654,413 U.S. Patent 5,658,734 U.S. Patent 5,661,028 U.S. Patent 5,665,547 U.S. Patent 5,667,972 U.S. Patent 5,670,663 U.S. Patent 5,672,697 U.S. Patent 5,677,195 U.S. Patent 5,681,947 U.S. Patent 5,695,940 U.S. Patent 5,700,637 U.S. Patent 5,700,922 U.S. Patent 5,705,629 U.S. Patent 5,708,153 U.S. Patent 5,708,154 U.S. Patent 5,714,606 U.S. Patent 5,728,525 U.S. Patent 5,728,525 U.S. Patent 5,744,305 U.S. Patent 5,763,167 U.S. Patent 5,770,358 U.S. Patent 5,777,092 U.S. Patent 5,789,162 U.S. Patent 5,792,847 U.S. Patent 5,800,992 U.S. Patent 5,807,522 U.S. Patent 5,830,645 U.S. Patent 5,837,196 U.S. Patent 5,847,219 U.S. Patent 5,856,174 U.S. Patent 5,858,988 U.S. Patent 5,859,221 U.S. Patent 5,871,928 U.S. Patent 5,872,232 U.S. Patent 5,876,932 U.S. Patent 5,886,165 U.S. Patent 5,919,626 U.S. Patent 5,922,591 U.S. Patent 6,004,755 U.S. Patent 6,040,193 U.S. Patent 6,087,102 U.S. Patent 6,251,666 U.S. Patent 6,368,799 U.S. Patent 6,383,749 U.S. Patent 6,617,112 U.S. Patent 6,638,717 U.S. Patent 6,720,138 U.S. Patent 6,723,509 U.S. Patent Seria109/545,207 U.S. Patent Serial 10/667,126 U.S. Patent Serial 11/141,707 U.S. Prov. Appln. 60/575,743 U.S. Prov. Appln. 60/649,584 U.S. Prov. Appln. Methods and Compositions Involving miRNA and miRNA Inhibitor Molecules, 2005 EP 266,032 PCT Appln. WO 0168255 PCT Appln. WO 03020898 PCT Appln. WO 03022421 PCT Appln. WO 03023058 PCT Appln. WO 03029485 PCT Appln. WO 03040410 PCT Appln. WO 03053586 PCT Appln. WO 03066906 PCT Appln. WO 03067217 PCT Appin. WO 03076928 PCT Appln. WO 03087297 PCT Appln. WO 03091426 PCT Appln. WO 03093810 PCT Appln. WO 03100448A1 PCT Appln. WO 04020085 PCT Appln. WO 04027093 PCT Appln. WO 09923256 PCT Appln. WO 09936760 PCT Appln. WO 93/17126 PCT Appln. WO 95/11995 PCT Appln. WO 95/21265 PCT Appln. WO 95/21944 PCT Appln. WO 95/35505 PCT Appln. WO 96/31622 PCT Appln. WO 97/10365 PCT Appln. WO 97/27317 PCT Appln. WO 9743450 PCT Appln. WO 99/35505 PCT Appln. WO0138580 PCT Appln. W003100012 UK Patent 1,529,202 UK Patent 8 803 000 Abuharbeid et al., Int JBiochem Cell Biol, 38(9):1463-8, 2006.
Akiba et al., Int J Oncol, 18(2):257-264, 2001.
Akino et al., Gastroenterology, 129(1):156-169, 2005.
Alevizos et al., Oncogene, 20(43):6196-6204, 2001.
Ambros, Cell, 107(7):823-826, 2001.
Ando et al., JBiol Chem, 279(24):25549-61, 2004.
Aplin et al., Br J Cancer, 74(2):240-245, 1996.
Ariyanayagam-Baksh et al., Mod Pathol, 16(10):992-995, 2003.
Ashburner et al., Nat Genet, 25(1):25-29, 2000.
Bagga et al., Cell, 122(4):553-563, 2005.
Bandyopadhyay et al., Oncogene, 21(22):3541-3551, 2002.
Bar-Shira et al., Cancer Res, 62(23):6803-6807, 2002.
Barton et al., Clin Cancer Res, 3(9):1579-1586, 1997.
Baumforth et al., Blood, 106(6):2138-2146, 2005.
Begum et al., Hepatology, 22(5):1447-1455, 1995.
Ben-Ze'ev and Raz, Cancer Res, 45(6):2632-2641, 1985.
Birchmeier et al., Nat Rev Mol Cell Biol, 4(12):915-25, 2003.
Bitomsky et al., Oncogene, 23(45):7484-93, 2004.
Blobe et al., JBiol Chem, 276(27):24627-37, 2001.
Boccaccio et al., Nat Rev Cancer, 6(8):637-45, 2006.
Bodner-Adler et al., Anticancer Res, 21(1 B):809-812, 2001.
Borczuk et al., Am J Pathol, 163(5):1949-1960, 2003.
Bos, Mutat Res, 195(3):255-271, 1988.
Bossy-Wetzel et al., Genes Dev, 6(12A):2340-2351, 1992.
Bravou et al., Int J Oncol, 27(6):1511-1518, 2005.
Brennecke et al., Cell, 113(1):25-36, 2003.
Budhu et al., Cancer Cell, 10(2):99-111, 2006.
Byrd et al., Blood,100:4325-4336, 2002.
Cahill et al., Nature, 392(6673):300-303, 1998.
Calin et al., Proc. Natl. Acad. Sci. USA, 99(24):15524-15529, 2002.
Camassei et al., Invest Ophthalmol Vis Sci, 44(6):2399-2403, 2003.
Carrano et al., Nat Cell Biol, 1(4):193-9, 1999.

Carrington and Ambros, Science, 301(5631):336-338, 2003.
Caselitz et al., Virchows Arch A Pathol Anat Histopathol, 400(1):43-51, 1983.
Chan et al., Appl Inununohistochem Mol Morphol, 10(4):322-326, 2002.
Chang et al., Int J Cancer, 114(6):942-949, 2005a.
Chang et al., Oncol Rep, 13(3):433-438, 2005b.
Chen et al., JPathol, 200(5):640-6, 2003.
Chieffi et al., Prostate, 66(3):326-33, 2006.
Christiansen et al., Leukemia, 17(9):1813-1819, 2003.
Chu et al., Cancer Res, 65(12):5301-5309, 2005.
Churg Am J Surg Pathol, 9(5):360-5, 1985.
Cipriano and Chen, Oncogene, 17(12):1549-1556, 1998.
Cooper et al., Nature, 311(5981):29-33, 1984.
Costello et al., Cancer Res, 57(7):1250-1254, 1997.
Cummins et al., In: IRT: Nucleosides and nucleosides, La Jolla CA, 72, 1996.
Danilkovitch-Miagkova et al., J Clin Invest, 109(7):863-7, 2002.
Davalos et al., Oncogene, 26(2):308-311, 2007.
Dean et al., Nature, 318(6044):385-8, 1985.
Demunter et al., Cancer Res, 61(12):4916-4922, 2001.
Denli et al., Trends Biochem. Sci., 28:196, 2003.
Didenko, Biotechniques, 31(5):1106-1116, 1118, 1120-1121, 2001.
Eferl et al., Cell, 112(2):181-192, 2003.
Egilmez et al., J Exp Clin Cancer Res, 20(4):549-552, 2001.
Egloff et al., Cancer Res, 66(1):6-9, 2006.
Einama et al., Pancreas, 32(4):376-381, 2006.
Emptage et al., Neuron, 29(1):197-208, 2001.
Endoh et al., Br J Cancer, 93(12):1395-1399, 2005.
Eng et al., Nat Rev Cancer, 3(3):193-202, 2003.
Esquela-Kerscher and Slack, Nat Rev Cancer, 6(4):259-269, 2006.
Ezzat et al., Clin Cancer Res, 11(3):1336-1341, 2005.
Ferrari et al., Cancer Res, 50(7):1988-1991, 1990.
Fesik, Nat Rev Cancer, 5(11):876-885, 2005.
Firth and Baxter, Endocr Rev, 23(6):824-854, 2002.
Fodor et al., Science, 251:767-777, 1991.
Froehler et al., Nucleic Acids Res., 14(13):5399-5407, 1986.

Furre et al., Cancer Res, 65(23):11051-11060, 2005.
Gao et al., Oncol Rep, 17(1):123-8, 2007.
Gerosa et al., JNatl Cancer Inst, 81(1):63-67, 1989.
Gilles et al., JPathol, 180(2):175-80, 1996.
Ginestier et al., Clin Cancer Res, 12(15):4533-4544, 2006.
Goke et al., Am JPhysiol Cell Physiol, 287(6):C1541-6, 2004.
Golay et al., Blood, 87(5):1900-1911, 1996.
Goldstone et al., Blood, 98(5):1302-11, 2001.
Gonzalez et al., Nature, 440(7084):702-6, 2006.
Grabsch et al., JPathol, 200(1):16-22, 2003.
Graff and Zimmer, Clin Exp Metastasis, 20(3):265-273, 2003.
Griffey et al., J. Mass Spectrom, 32(3):305-13, 1997.
Grimwade, Best.Pract.Res. Clin.Haematol., 14:497-529, 2001.
Grosshans et al., Dev Cell, 8(3):321-330, 2005.
Gstaiger et al., Proc Natl Acad Sci USA, 98(9):5043-8, 2001.
Guo et al., Carcinogenesis, 27(3):454-464, 2006.
Halaban et al., J Exp Med, 191(6):1005-1016, 2000.
Han et al., J Recept Signal Transduct Res, 23(2-3):225-238, 2003.
Hanahan and Weinberg, Cell 100(1):57-70, 2000.
Hansel et al., Am J Surg Pathol, 29(3):390-399, 2005.
Hardwick et al., Cancer Res, 59(4):831-842, 1999.
Hartmann et al., Cancer Res, 59(7):1578-1583, 1999.
Hayette et al., Blood, 102(4):1549-1550, 2003.
He et al., Nature, 435(7043):828-833, 2005.
He et al., Proc. Natl. Acad. Sci. USA, 102(52):19075-19080, 2005.
Hiddemann et al., J. Clin. Oncol./ 17(11): 3569-76, 1999.
Hishikawa et al., JBiol Chem, 274(52):37461-6, 1999.
Hu et al., Oncogene, 23(1):298-302, 2004.
Huang et al., Clin Cancer Res, 12(2):487-498, 2006.
Huusko et al., Nat Genet, 36(9):979-983, 2004.
Hynes et al., Nat Rev Cancer, 5(5):341-54, 2005.
Inui et al., Biochem Biophys Res Commun, 303(3):978-984, 2003.
Islam et al., J Cell Biochem, 78(l):141-50, 2000.
Itakura and Riggs, Science, 209:1401-1405, 1980.

Ito et al., Anticancer Res, 23(3B):2335-2338, 2003.
Ito et al., Anticancer Res, 25(5):3419-3423, 2005.
Jaakkola et al., Int J Cancer, 54(3):378-382, 1993.
Jansen et al., Cancer Res, 65(14):6034-41, 2005.
Jansen et al., Mol Cancer Ther, 3(2):103-10, 2004.
Jiang et al., Cancer Res, 64(16):5787-5794, 2004.
Johnson et al., Cell, 120:635-647, 2005.
Jubb et al., Clin Cancer Res, 11(14):5181-5187, 2005.
Jung et al., Pathol Int, 56(9):503-509, 2006.
Kamata et al., J Cancer Res Clin Oncol, 131(9):591-596, 2005.
Karakaidos et al., Am JPathol, 165(4):1351-1365, 2004.
Kawagoe et al., Cancer Res, 57(12):2516-2521, 1997.
Keen and Taylor, Nat Rev Cancer, 4(12):927-936, 2004.
Khoury et al., Ann Diagn Pathol, 6(3):154-158, 2002.
Killion et al., BMC. Bioinformatics, 4:32, 2003.
Kim et al., Clin Cancer Res, 11(2 Pt 1):473-482, 2005.
Kishi et al., JBiol Chem, 281(25):17492-17500, 2006.
Kletsas et al., Biochem Pharmacol, 67(10):1927-1932, 2004.
Klostermeier and Millar, Biopolymers, 61(3):159-79, 2001-2002.
Kobayashi et al., Oncogene, 22(9):1294-1301, 2003.
Koivunen et al., Cancer Lett, 235(1):1-10, 2006.
Koivunen et al., Cancer Res, 64(16):5693-701, 2004.
Kokko et al., BMC Cancer, 6:145, 2006.
Komberg and Baker, In: DNA Replication, 2d Ed., Freeman, San Francisco, 1992.
Koumakpayi et al., Clin Cancer Res, 12(9):2730-2737, 2006.
Kristjansdottir and Rudolph, Chem Biol, 11(8):1043-1051, 2004.
Kuhajda Cancer Res, 66(12):5977-80, 2006.
Lacayo et al., Blood, 104(9):2646-2654, 2004.
Lagos-Quintana et al., Science, 294(5543):853-858, 2001.
Lahn and Sundell, Melanoma Res, 14(2):85-89, 2004.
Lam et al., Br JNeurosurg, 14(1):28-32, 2000.
Lambros et al., J Pathol, 205(1):29-40, 2005.
Lau et al., Science, 294(5543):858-862, 2001.
Lauffart et al., BMC Womens Health, 5:8, 2005.

Le Frere-Belda et al., Br J Cancer, 85(10):1515-1521, 2001.
Lee and Ambros, Science, 294(5543):862-864, 2001.
Lemoine et al., Br J Cancer, 66(6):1116-1121, 1992.
Leng et al., Chin Med Sci J, 12(2):67-70, 1997.
Lim et al., Nature, 433(7027):769-773, 2005 Lin et al., Dev Cell, 4(5):639-650, 2003.
Lin et al., Gastroenterology, 128(1):9-23, 2005.
Liu et al., Int J Hematol, 84(5):425-431, 2006.
Liu et al., Proc Natl Acad Sci USA, 100(10):5789-94, 2003.
Livak and Schrnittgen, Methods, 25:402-408, 2001.
Lu et al., Nature, 435(7043):834-838, 2005.
Maaser et al., Clin Cancer Res, 11(5):1751-1756, 2005.
Malumbres and Barbacid, NatRev Cancer, 1(3):222-231, 2001.
Mao etal., Blood, 101(4):1513-1519, 2003.
Marcucci et al., JClin.Oncol., 23:5705-5717, 2005.
Mariani Costantini et al., Cancer Res, 50(18):6107-6112, 1990.
Marone et al., .Int J Cancer, 75(l):34-39, 1998.
Martinez-Delgado et al., Br JHaematol, 109(1):97-103, 2000.
Mathas et al., Embo J, 21(15):4104-4113, 2002.
Maurer et al., Hum Pathol, 29(8):771-777, 1998.
McGary et al., Cancer Biol Ther, 1(5):459-465, 2002.
Mclnroy et al., Biochem Biophys Res Commun, 360(1):109-14, 2007.
Mendrzyk et al., J Clin Oncol, 23(34):8853-8862, 2005.
Menendez et al., Proc Natl Acad Sci U S A, 101(29):10715-10720, 2004.
Miyake et al., Cancer, 86(2):316-324, 1999.
Miyaki and Kuroki, Biochem Biophys Res Commun, 306(4):799-804, 2003.
Montero et al., Clin Cancer Res, 4(9):2161-2168, 1998.
Monti et al., Virchows Arch., 445(3):236-247, 2004.
Mossink et al., Oncogene, 22(47):7458-7467, 2003.
Mrozek et al., Blood, 06-001149v1, 2006.
Mundt et al., Biochem Biophys Res Commun, 239(2):377-85, 1997.
Murphy et al., J Clin Pathol, 58(5):525-534, 2005.
Nakada et al., Cancer Res, 64(9):3179-3185, 2004.
Neri et al., JExp Med, 170(5):1715-1725, 1989.

Ng et al., Blood, 89(7):2500-2506, 1997.
Ngan et al., Br J Cancer, 96(6):986-92, 2007.
Ohsaki et al., Cancer Ites. Jul 1;52(13):3534-8, 1992.
Olsen et al., Dev. Biol., 216:671, 1999.
Osthus et al., Cancer Res, 65(13):5620-5627, 2005.
Ott et al., Oncogene, 26(33):4863-4871, 2007.
Ovcharenko et al., RNA. Jun; l 1(6):985-93, 2005.
Oyama et al., Pathol Int, 45(1):45-50, 1995.
Paquette et al., Oncogene, 5(11):1659-1663, 1990.
Passegue et al., Cell, 119(3):431-443, 2004.
Payton and Coats, Int J Biochem Cell Biol, 34(4):315-320, 2002.
Payton et al., Oncogene, 21(55):8529-8534, 2002.
Petit et al., Genomics, 57(3):438-441, 1999.
Pretner et al., Anticancer Res, 26(lA):9-22, 2006.
Prochownik et al., Genes Chromosomes Cancer, 22(4):295-304, 1998.
Qian et al., Proc Natl Acad Sci U S A, 99(23):14925-14930, 2002.
Rabinovitz et al., Clin Exp Metastasis, 13(6):481-491, 1995.
Rajkumar et al., J Pathol, 179(4):381-385, 1996.
Ree et al., Cancer Res, 59(18):4675-4680, 1999.
Reinhart et al., Nature, 403(6772):901-906, 2000.
Reiter et al., Clin Cancer Res, 12(17):5136-5141, 2006 Robles et al., JBiol Chem, 277(28):25431-25438, 2002.
Rosenkilde et al., Apmis, 112(7-8):481-95, 2004.
Ru et al., Oncogene, 21(30):4673-4679, 2002.
Rutka et al., Int J Dev Neurosci, 17(5-6):503-515, 1999.
Saigusa et al., Cancer Sci, 96(10):676-683, 2005.
Sambrook et al., In: DNA microaarays: a molecular cloning manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, 2001, 2003.
Schaich et al., Br. J. Haematol., 112:300-307, 2001.
Scheit, In: Synthesis and Biological Function, Wiley-Interscience, New York, 172, 1980.
Seggerson et al., Dev. Biol., 243:215, 2002.
Seike et al., Lung Cancer, 38(3):229-234, 2002.
Semple et al., Biotechnol Adv, 22(8):621-31, 2004.

Serrano et al., Acta Haematol, 116(2):77-89, 2006.
Shah et al., Oncogene, 21(54):8251-8261, 2002.
Shapira et al., Cancer, 103(7):1336-1346, 2005.
Shetty et al., Br J Cancer, 93(11):1295-1300, 2005.
Shi et al., Cancer Res, 51(10):2690-2693, 1991.
Shibahara et al., Anticancer Res, 25(3B): 1881-1888, 2005.
Shigeishi et al., Oncol Rep, 15(4):933-938, 2006.
Shimoyama et al., Clin Cancer Res, 5(5):1125-1130, 1999.
Singh et al., Cancer Res, 63(9):2306-2311, 2003.
Slack et al., Molec Cell, 5(4):659-669, 2000.
Slotky et al., Breast Cancer Res, 7(5):R737-744, 2005.
Smith et al., Biochem Biophys Res Commun, 234(2):397-405, 1997.
Smith et al., Br J Cancer, 93(6):719-29, 2005.
Sommers et al., Cancer Res, 52(19):5190-5197, 1992.
Soufla et al., Cancer Lett, 221(1):105-118, 2005.
Sparmann et al., Cancer Cell, 6(5):447-58, 2004.
Stark et al., Blood, 63(2):415-420, 1984.
Steensma et al., Blood, 105(2):443-452, 2005.
Strebhardt et al., Nat Rev Cancer, 6(4):321-30, 2006.
Su et al., Clin Cancer Res, 7(5):1320-1324, 2001.
Sui et al., Oncol Rep, 15(4):765-771, 2006.
Sutter et al., Int J Cancer, 102(4):318-327, 2002.
Takamizawa et al., Cancer Res, 64(11):3753-3756, 2004 Takanami, Oncol Rep, 13(4):727-731, 2005.
Tanner et al., Clin Cancer Res, 6(5):1833-1839, 2000.
Tassi et al., Cancer Res, 66(2):1191-1198, 2006.
Tassi et al., JBiol Chem, 276(43):40247-53, 2001.
Teofili et al., Leukemia, 17(5):919-924, 2003.
Traub et al., Breast Cancer Res Treat, 99(2):185-191, 2006.
Tsai et al., J Natl Cancer Inst. Jun 2;85(11):897-901, 1993.
Turner et al., Nat Rev Cancer, 4(10):814-9, 2004.
Uemura et al., Int J Mol Med, 18(2):365-373, 2006.
Ulisse et al., Int J Cancer, 119(2):275-282, 2006.
Upton et al., Am J Surg Pathol, 10(8):560-7, 1986.

Vella et al., Chem Biol, 11(12):1619-1623, 2004b.
Vella et al., Genes Dev, 18(2):132-7, 2004a.
Venkatasubbarao et al., Anticancer Res, 20(lA):43-51, 2000.
Vlodavsky and Soustiel, J Neurooncol, 81(1):1-7, 2007.
Vos et al., JBiol Chem, 278(30):28045-51, 2003.
Wang et al., Mol Carcinog, 31(2):90-100, 2001.
Wang et al., Oncogene, 22(10):1486-1490, 2003.
Wechsler et al., Cancer Res, 57(21):4905-4912, 1997.
Weichert et al., Int J Oncol, 23(3):633-639, 2003.
Weil et al., Biotechniques. 2002 Dec;33(6):1244-8, 2002.
Weiss and Bohmann, Cell Cycle, 3(2):111-113, 2004.
Wewer et al., Am JPathol, 151(5):1191-1198, 1997.
Wickborn et al., Mol Carcinog, 45(8):572-581, 2006.
Wilcoxon, Biometrics, 1:80-83, 1945.
Wohlschlegel et al., Am JPathol, 161(1):267-273, 2002.
Wolowiec et al., Leuk Lymphoma, 35(1-2):147-157, 1999.
Wooster and Weber, NEngl JMed, 348(23):2339-2347, 2003.
Woszczyk et al., Med Sci Monit, 10(1):CR33-37, 2004.
Wu et al., Gynecol Oncol, 102(1):15-21, 2006.
Wu et al., Pathol Oncol Res, 10(1):26-33, 2004.
Xu et al., Curr. Biol., 13(9):790-795, 2003.
Xue et al., Cancer Res, 66(3):1418-1426, 2006.
Yamamoto et al., Int J Oncol, 13(2):233-239, 1998.
Yang et al., Am JRespir Cell Mol Biol, 21(2):216-222, 1999.
Yang et al., Blood, 101(8):3205-3211, 2003.
Yang et al., J Androl, 22(3):471-480, 2001.
Yang et al., Mol Cell Biol, 23(1):26-37, 2003.
Yang et al., Mol Cell Biol, 26(4):1297-306, 2006.
Yano et al., Int JMol Med, 12(5):763-766, 2003.
Yasui et al., Hepatology, 35(6):1476-1484, 2002.
Ye et al., Biochim Biophys Acta, 1493(3):373-377, 2000.
Zhang et al., Chin Med J(Engl), 112(4):330-332, 1999.
Zhang et al., Oncogene, 25(45):6101-12, 2006.

Claims (58)

1. A method of modulating gene expression in a cell comprising administering to the cell an amount of an isolated nucleic acid comprising a let-7 nucleic acid sequence in an amount sufficient to modulate the expression of a gene modulated by a let-7 miRNA family member.

2. The method of claim 1, wherein the gene modulated comprises one or more gene identified in Table 2, 3, 4, 5, 6, 7, 8, and 9.

3. The method of claim 2, wherein the expression of a gene is down-regulated.

4. The method of claim 2, wherein the expression of a gene is up-regulated.

5. The method of claim 2, wherein the gene modulated comprises one or more of ATRX, AURKA/STK6, AURKB/STK12, BRCA1, BRCA2, BUB1, BUB1B, BZRP, CCNA2, CCNB1, CCNE2, CCNG2, CDC2, CDC20, CDC23, CDC25A, CDC6, CDCA7, CDK2, CDK6, CDKN2B, CDT1, CEBPD, CKS1B, CSF1, EIF4E, EPHB2, ERBB3, FASN, FGFBPI, FGFR4, FH, GMNN, IGFBP1, IL8, ITGA6, JUN, JUNB, LHFP, MCAM, MET, MVP, MXI1, MYBL1, MYBL2, NRAS, P8, PDCD4, PLK1, PRKCA, RASSF2, SIVA, SKP2, SMAD4, TACC3, TFDP1, TGFBR3, TNFSF10, or VIM.

6. The method of claim 5, wherein the genes modulated are ATRX, AURKA/STK6, AURKB/STK12, BRCA1, BRCA2, BUB1, BUB1B, BZRP, CCNA2, CCNB1, CCNE2, CCNG2, CDC2, CDC20, CDC23, CDC25A, CDC6, CDCA7, CDK2, CDK6, CDKN2B, CDT1, CEBPD, CKS1B, CSF1, EIF4E, EPHB2, ERBB3, FASN, FGFBP1, FGFR4, FH, GMNN, IGFBP1, IL8, ITGA6, JUN, JUNB, LHFP, MCAM, MET, MVP, MXI1, MYBL1, MYBL2, NRAS, P8, PDCD4, PLK1, PRKCA, RASSF2, SIVA, SKP2, SMAD4, TACC3, TFDP1, TGFBR3, TNFSF10, and VIM.

7. The method of claim 1, wherein the cell is in a subject having, suspected of having, or at risk of developing a metabolic, an immunologic, an infectious, a cardiovascular, a digestive, an endocrine, an ocular, a genitourinary, a blood, a musculoskeletal, a nervous system, a congenital, a respiratory, a skin, or a cancerous disease or condition.

8. The method of claim 8, wherein the infectious disease or condition is a parasitic, bacterial, viral, or fungal infection.

9. The method of claim 7, wherein the cell is in a subject having, suspected of having, or at risk of developing acute lymphocytic leukemia; acute myeloid leukemia;
alpha thalassemia; angiosarcoma; astrocytoma; breast carcinoma; bladder carcinoma;
Burkitt's lymphoma; cervical carcinoma; carcinoma of the head and neck;
chronic lymphocytic leukemia; chronic myeloblastic leukemia; colorectal carcinoma;
endometrial carcinoma; fibrosarcoma glioma; glioblastoma; glioblastoma multiforme;
gastric carcinoma; gastrinoma; hepatoblastoma; hepatocellular carcinoma;
Hodgkin lymphoma; Kaposi's sarcoma; larynx carcinoma; leukemia; lung carcinoma;
leiomyoma; leiomyosarcoma; lipoma; melanoma; medulloblastoma; myeloid leukemia; mesothelioma; myxofibrosarcoma; multiple myeloma; neuroblastoma; non-Hodgkin lymphoma; non small cell lung carcinoma; ovarian carcinoma; esophageal carcinoma; oropharyngeal carcinoma; osteosarcoma; pancreatic carcinoma;
papillary carcinoma; prostate carcinoma; promyelocytic leukemia; renal cell carcinoma;
retinoblastoma; rhabdomyosarcoma; sporadic papillary renal carcinoma; squamous cell carcinoma of the head and neck; salivary gland tumor; small intestinal carcinoma;
T-cell leukemia; thyroid carcinoma; or orurothelial carcinoma, wherein the modulation of one or more gene is sufficient for a therapeutic response.

10. The method of claim 9, wherein a therapeutic response is reduced viability, reduced proliferation, or reduced metastasis of a cancer cell.

11. The method of claim 1, wherein the let-7 nucleic acid comprises at least one of hsa-1et-7a-1, hsa-1et-7a-2, hsa-1et-7a-3, hsa-1et-7b, hsa-1et-7c, hsa-1et-7d, hsa-1et-7e, hsa-1et-7f-1, hsa-1et-7f-2, hsa-1et-7g, hsa-1et-7i, or a segment thereof.

12. The method of claim 1, wherein the let-7 nucleic acid is an inhibitor of 1et-7 function.

13. The method of claim 1, wherein the cell is an epithelial, a stromal, or a mucosal cell.

14. The method of claim 1, wherein the cell is a brain, a neuronal, a blood, a cervical, an esophageal, a lung, a cardiovascular, a liver, a breast, a bone, a thyroid, a glandular, an adrenal, a pancreatic, a stomach, a intestinal, a kidney, a bladder, a prostate, a uterus, an ovarian, a testicular, a splenic, a skin, a smooth muscle, a cardiac muscle, a striated muscle cell.

15. The method of claim 1, wherein the cell is a cancer cell.

16. The method of claim 15, wherein the cancer cell is a neuronal, glial, lung, liver, brain, breast, bladder, blood, leukemic, colon, endometrial, stomach, skin, ovarian, fat, bone, cervical, esophageal, pancreatic, prostate, kidney, uterine, testicular, epithelial, muscle, oropharyngeal, adrenal, gastrointestinal, mesothelial, salivary gland, small intestine, or thyroid cell.

17. The method of claim 1, wherein the isolated let-7 nucleic acid is a recombinant nucleic acid.

18. The method of claim 17, wherein the recombinant nucleic acid is RNA.

19. The method of claim 17, wherein the recombinant nucleic acid is DNA.

20. The method of claim 19, wherein the recombinant nucleic acid comprises a 1et-7 expression cassette.

21. The method of claim 20, wherein the expression cassette is comprised in a viral vector, or plasmid DNA vector.

22. The method of claim 21, wherein the viral vector is administered at a dose of 1x10 5 to 1x10 14 viral particles per dose or the plasmid DNA vector is administered at a dose of 100 mg per patient to 4000 mg per patient.

23. The method of claim 1, wherein the let-7 nucleic acid is a synthetic nucleic acid.

24. The method of claim 23, wherein the nucleic acid is administered at a dose of 0.01 mg/kg of body weight to 10 mg/kg of body weight.

25. The method of claim 1, wherein the 1et-7 is a hsa- 1et-7.

26. The method of claim 1, wherein the nucleic acid is administered enterally or parenterally.

27. The method of claim 26, wherein enteral administration is orally.

28. The method of claim 26, wherein parenteral administration is intravascular, intracranial, intrapleural, intratumoral, intraperitoneal, intramuscular, intralymphatic, intraglandular, subcutaneous, topical, intrabronchial, intratracheal, intranasal, inhaled, or instilled.

29. The method of claim 1, wherein the nucleic acid is comprised in a pharmaceutical formulation.

30. The method of claim 29, wherein the pharmaceutical formulation is a lipid composition.

31. The method of claim 29, wherein the pharmaceutical formulation is a nanoparticle composition.

32. The method of claim 29, wherein the pharmaceutical formulation consists of biocompatible and/or biodegradable molecules.

33. A method of modulating a cellular pathway or a physiologic pathway comprising administering to a cell an amount of an isolated nucleic acid comprising a 1et-7 nucleic acid sequence in an amount sufficient to modulate the cellular pathway or physiologic pathway that includes one or more genes identified or gene products related to one or more genes identified in Table 2, 3, 4, 5, 6, 7, 8 and 9.

34. The method of claim 33, wherein the gene modulated comprises one or more of ATRX, AURKA/STK6, AURKB/STK12, BRCA1, BRCA2, BUB1, BUB1B, BZRP, CCNA2, CCNB1, CCNE2, CCNG2, CDC2, CDC20, CDC23, CDC25A, CDC6, CDCA7, CDK2, CDK6, CDKN2B, CDT1, CEBPD, CKS1B, CSF1, EIF4E, EPHB2, ERBB3, FASN, FGFBP1, FGFR4, FH, GMNN, IGFBP1, IL8, ITGA6, JUN, JUNB, LHFP, MCAM, MET, MVP, MXI1, MYBL1, MYBL2, NRAS, P8, PDCD4, PLK1, PRKCA, RASSF2, SIVA, SKP2, SMAD4, TACC3, TFDP1, TGFBR3, TNFSF10, or VIM.

35. The method of claim 33, wherein the let-7 nucleic acid comprises at least one of hsa-1et-7a-1, hsa-1et-7a-2, hsa-1et-7a-3, hsa-1et-7b, hsa-1et-7c, hsa-1et-7d, hsa-1et-7e, hsa-1et-7f-1, hsa-1et-7f-2, hsa-1et-7g, hsa-1et-7i, or a segment thereof.

36. The method of claim 33, further comprising administering 2, 3, 4, 5, 6, or more miRNAs.

37. The method claim 36 wherein the miRNAs are comprised in a single composition.

38. The method of 33, wherein at least two cellular pathways or physiologic pathways are modulated.

39. The method of claim 36, wherein at least one gene is modulated by multiple miRNAs.

40. The method of claim 33, wherein the expression of a gene or a gene product is down-regulated.

41. The method of claim 33, wherein the expression of a gene or a gene product is up-regulated.

42. The method of claim 33, wherein the cell is a cancer cell.

43. The method of claim 42, wherein the cancer cell is a lung, liver or acute myeloid leukemic cancer cell.

44. The method of claim 42, wherein viability of the cell is reduced, proliferation of the cell is reduced, metastasis of the cell is reduced, or the cell's sensitivity to therapy is increased.

45. The method of claim 42, wherein the cancer cell is neuronal, glial, lung, liver, brain, breast, bladder, blood, leukemic, colon, endometrial, stomach, skin, ovarian, fat, bone, cervical, esophageal, pancreatic, prostate, kidney, uterine, testicular, epithelial, muscle, oropharyngeal, adrenal, gastrointestinal, mesothelial, or thyroid cell.

46. The method of claim 33, wherein the isolated let-7 nucleic acid is a recombinant nucleic acid.

47. The method of claim 46, wherein the recombinant nucleic acid is DNA.

48. The method of claim 47, wherein the recombinant nucleic acid is a viral vector or a plasmid DNA.

49. The method of claim 33, wherein the nucleic acid is RNA.

50. The method of claim 46, wherein the recombinant nucleic acid is a synthetic nucleic acid.

51. A method of treating a patient diagnosed with or suspected of having or suspected of developing a pathological condition or disease related to a gene modulated by a miRNA comprising the steps of:
(a) administering to the patient an amount of an isolated nucleic acid comprising a let-7 nucleic acid sequence in an amount sufficient to modulate a cellular pathway or a physiologic pathway; and (b) administering a second therapy, wherein the modulation of the cellular pathway or physiologic pathway sensitizes the patient to the second therapy.

52. The method of claim 51, wherein one or more cellular pathway or physiologic pathway includes one or more genes identified in Table 2, 3, 4, 5, 6, 7, 8 and 9.

53. A method of selecting a miRNA to be administered to a subject with, suspected of having, or having a propensity for developing a pathological condition or disease comprising:

(a) determining an expression profile of one or more genes selected from Table 2, 3, 4, 5, 6, 7, 8 and 9;
(b) assessing the sensitivity of the subject to miRNA therapy based on the expression profile; and (c) selecting one or more miRNA based on the assessed sensitivity.

54. The method of claim 53 further comprising treating the subject with 1, 2, 4, 5, 6, 7, 8, 9, 10, or more miRNAs.

55. The method of claim 54, wherein each miRNA is administered individually or one or more combinations.

56. The method of claim 55, wherein the miRNAs are in a single composition.

57. A method of assessing a cell, tissue, or subject comprising assessing expression of let-7 in combination with assessing expression of one or more gene from Table 2, 3, 4, 5, 6, 7, or 8 in at least one sample.

58. A method of assessing 1et-7 status in a sample comprising the steps of:

(a) assessing expression of one or more genes from Table 2, 3, 4, 5, 6, 7, 8 or 9 in a sample; and (b) determining 1et-7 status based on level of 1et-7 expression in the sample.